The Role of RING Finger Proteins in Chromatin Remodeling and Biological Functions

Abstract

Mammalian DNA duplexes are highly condensed with different components, including histones, enabling chromatin formation. Chromatin remodeling is involved in multiple biological processes, including gene transcription regulation and DNA damage repair. Recent research has highlighted the significant involvement of really interesting new gene (RING) finger proteins in chromatin remodeling, primarily attributed to their E3 ubiquitin ligase activities. In this review, we highlight the pivotal role of RING finger proteins in chromatin remodeling and provide an overview of their capacity to ubiquitinate specific histones, modulate ATP-dependent chromatin remodeling complexes and interact with various histone post-translational modifications. We also discuss the diverse biological effects of RING finger protein-mediated chromatin remodeling and explore potential therapeutic strategies for targeting these proteins.

Keywords::

• chromatin remodeling
• DNA damage repair
• E3 ubiquitin ligase
• RING finger protein
• transcriptional regulation

Chromatin, the fundamental structure of eukaryotic cells, consists of DNA, histones and nonhistone proteins, and plays a crucial role in maintaining genetic stability, regulating gene expression and transmitting genetic information. Chromatin is classified into euchromatin and heterochromatin. The loosely arranged chromatin structure of euchromatin allows transcription factors and regulators easy access to DNA sequences, leading to enhanced transcription and gene expression. Conversely, heterochromatin is a condensed state of chromatin that inhibits gene transcription by limiting the access of transcription factors to DNA binding sites. Chromatin remodeling modulates gene expression by regulating the dynamic transition process between euchromatin and heterochromatin at specific developmental stages or cell states [1].

Nucleosomes occupy a central role in the organization of chromatin. The dynamic substance of chromatin packages and regulates the genetic material within the cell nucleus. At the core of the nucleosome, the fundamental structural unit consists of an octamer composed of four pairs of histones: histone H2A, histone H2B, histone H3 and histone H4. This octamer acts as a spool around which DNA is wrapped, creating a condensed chromatin structure. The interplay of nucleosomes and their associated proteins governs essential cellular processes by modulating access to the underlying DNA. The functional flexibility of chromatin is evident through numerous modifications that can be applied to its components. These modifications orchestrate a symphony of molecular events that facilitate chromatin remodeling and precisely regulate gene expression. These processes are mainly affected by DNA methylation, post-translational modifications (PTMs) of histone and nonhistone proteins, ATP-dependent chromatin remodeling complexes, and crosstalk among different types of histone modifications.

Histone PTMs play a key role in the regulation of chromatin. Histones undergo a wide array of PTMs, including methylation, acetylation, ubiquitination and phosphorylation. These chemical modifications serve as dynamic switches, affecting chromatin compaction and gene accessibility. In addition, the crosstalk between various types of histone PTMs increases the complexity of the regulatory landscape, whereby one modification can affect the placement or removal of others [2,3]. For instance, histone ubiquitination, the addition of ubiquitin molecules to histones, plays a crucial role in gene transcriptional regulation, DNA damage repair and the overall maintenance of chromatin integrity [4]. It acts as a signal for various cellular processes, highlighting the complex network of interactions within the chromatin environment. Thus, nucleosomes, with their histone octamer cores, constitute the fundamental building blocks of chromatin, and the intricate modifications applied to these components finely orchestrate gene regulation. Understanding the language of nucleosome modifications is crucial to unravel the complexity of cellular processes, ranging from gene expression to DNA repair.

Really interesting new gene (RING) finger proteins are the most commonly found E3 ubiquitin ligases that are characterized by the RING finger domain. In addition to promoting ubiquitin transfer from the ubiquitin-conjugating enzyme E2 to the target protein, RING finger proteins regulate protein degradation and participate in cellular processes such as cell cycle progression, signal transduction and DNA repair. RING E3 ubiquitin ligases can be categorized into different subtypes depending on their mechanisms of action and associated protein domains. RING finger protein E3 ligases, including NEDD4 [5], MDM2 [6] and TRIM proteins [7], independently and directly interact with target proteins. RING finger protein E3 ligase-containing complexes provide substrate recognition and binding functions, including in complexes such as APC/C and SCF. By elucidating the role of RING E3 ligases, we can achieve a better understanding of their biological functions and design more effective therapeutic interventions for cancers and autoimmune diseases [8].

In this review, we summarize the modulation of RING finger proteins in chromatin remodeling by ubiquitinating different histones, regulating ATP-dependent chromatin remodeling complexes and engaging with other histone PTMs. Furthermoew, we discuss the diverse biological effects caused by RING finger protein-regulated chromatin remodeling and potential therapeutic approaches to RING finger protein targeting.

RING finger proteins ubiquitinate histones as E3 ubiquitin ligases

RING finger proteins mediate histone ubiquitination through their ubiquitin ligase activity, modulating chromatin structure and regulating diverse biological processes. Histone ubiquitination occurs primarily at specific histones, which has distinct implications for gene regulation [9,10], DNA replication [11], DNA damage repair [12,13] and chromatin structure [14–16]. We have summarized RING finger proteins ubiquitinating different lysine (Lys) sites of core histones (Figure 1&Table 1).

Figure 1. The nucleosome octameric structure consists of four core histone pairs: H2A, H2B, H3 and H4.

As E3 ubiquitin ligases, RING finger proteins ubiquitinate different Lys (K) sites of histones. The RING finger proteins corresponding to the ubiquitinated histone Lys sites are shown. Also see also Table 1 for references.

K: Lysine; Ub: Ubiquitin; Unknown: Lysine site remains unknown.

Table 1. RING finger proteins ubiquitinate different lysine sites of core histones.

RING finger protein	Histone lysine site	Ref.
	H2A
PRC1	H2AK119Ub	[17]
hPRC1L	H2AK119Ub	[18]
BCOR	H2AK119Ub	[19]
PCGF2/Mel-18-RING1B	H2AK119Ub	[20]
PCGF6	H2AK119Ub	[21]
KDM2B-PCGF1-RING1B	H2AK119Ub	[22,23]
BMI1-RING1A/B	H2AK118/119Ub	[24,25]
RNF8	H2AK13-15Ub	[26]
RNF168	H2AK13-15Ub	[26,27]
BRCA1/BARD1	H2AK124/127/129Ub	[28–36]
TRIM37	H2AK (unknown)	[7]
DZIP3 (hRUL138)	H2AK119Ub	[37]
FRRUC	H2AK119Ub	[23]
UBR1/UBR2	H2A (unknown)	[38,39]
PHF7	H2A (unknown)	[40]
DTX3L	H2AK (unknown)	[41]
HUWE1 (LASU1)	H2AK (unknown)	[42]
SHPRH	H2AK (unknown)	[43]
	H2B
RNF20/40	H2BK120	[9,10,44]
PHF6	H2BK120	[45]
MDM2	H2BK120	[46]
MSL1/MSL2	H2BK34	[47,48]
BRCA1/BARD1	H2BK (unknown)	[36]
DTX3L	H2BK (unknown)	[41]
HUWE1 (LASU1)	H2BK (unknown)	[42]
SHPRH	H2BK (unknown)	[43]
	H3
CLRC	H3K14	[49]
PHF7	H3K14	[50]
UHRF1	H3K18/23Ub	[11,51]
NEDD4	H3K23/36/37	[5]
CRL4	H3K79	[52]
BRCA1/BARD1	H3K (unknown)	[36]
HUWE1 (LASU1)	H3K (unknown)	[42]
MSL1-MSL2	H3K (unknown)	[47]
RNF4	H3K (unknown)	[53]
RNF138	H3K (unknown)	[54]
SHPRH	H3K (unknown)	[43]
CUL4-DDB-ROC1/RBX1	H3K (unknown)	[16]
	H4
Cul4A	H4K31	[55]
BBAP	H4K91	[56]
DTX3L	H4K (unknown)	[57]
BRCA1/BARD1	H4K (unknown)	[36]
HUWE1 (LASU1)	H4K (unknown)	[42]
MSL1-MSL2	H4K (unknown)	[47]
SHPRH	H4K (unknown)	[43]
CUL4-DDB-ROC1/RBX1	H4K (unknown)	[16]

Regulation of histone H2A ubiquitination by RING finger proteins

Histone H2A ubiquitination contributes to gene silencing or repression. One well-known form of histone H2A ubiquitination is the monoubiquitination of Lys-119 (H2AK119Ub). H2AK119Ub promotes chromatin compaction and recruits additional chromatin modifiers as a transcriptionally repressive mark for gene repression [24]. RING domain-containing E3 ubiquitin ligases are the primary catalytic enzymes for histone H2A ubiquitination. RING1B (RING2, encoded by RNF2) is essential to histone modification and gene regulation by facilitating H2AK119Ub mediation [18,24,58–61]. PRC1, an E3 core subunit of RING1A/B, monoubiquitinates histone H2AK119 by forming a complex with the E2 enzyme UbcH5c [62,63], which is involved in chromatin inactivation [17] and silencing of HOX family genes [18,24,64]. In addition, hPRC1L, containing RING1A and RING1B, monoubiquitinates nucleosomal histone H2AK119, resulting in transcriptional repression of drosophila Ubx [18]. BCOR, consisting of RING1B, mediates histone H2AK119 monoubiquitination, further inhibiting specific transcription targets such as HOXA cluster genes, inducing heritable changes in chromatin [65].

In addition, the PCGF2–RING1B complex, composed of two ubiquitin E3 ligases, RING1B and PCGF2 (also known as RNF110), significantly enhances ubiquitin ligase activity through nonspecifically ubiquitylating free histone substrates and highly specifically targeting histone H2A Lys-119 within nucleosomes. Mutation analysis indicates that RING1B is necessary to perform the E3 function, but PCGF2 directs this activity toward histone H2AK119 on chromatin [20]. The RING finger protein 2A-HUB (also known as RING-type E3 ubiquitin transferase DZIP3) catalyzes histone H2AK119 monoubiquitination [66], further repressing a subset of chemokine gene expression.

In addition to Lys-119, histone H2A ubiquitination can occur at other Lys residues. For example, BRCA1 facilitates the formation of polyubiquitin chains linked by Lys-6. The E3 ligase activity of the BRCA1–BARD1 complex is significantly enhanced as a result of the heterodimerization between BRCA1 and BARD1 [67]. BRCA1–BARD1 ubiquitinates histone H2A specifically at Lys residues 127–129 in vitro and in vivo, causing further silencing at DNA satellite repeat regions by accumulating ubiquitin conjugate complex at DNA damage sites [28]. RNF8 and RNF168 modulate chromatin structure by ubiquitinating histone H2A at Lys-13/Lys-15 [26,68]. TRIM37 is a novel histone H2A ubiquitin ligase that mediates the binding of PRC2 to specific target genes, such as Fas [7], with unknown specific Lys sites. Furthermore, the RING-type E3 ligases UBR1 and UBR2 facilitate either the monoubiquitination or polyubiquitination of histone H2A in vitro [38], contributing to transcriptional repression during cell meiosis and chromatin inactivation [39], although the precise Lys site at which UBR2 ubiquitinates histone H2A remains unknown.

Regulation of histone H2B ubiquitination by RING finger proteins

RING finger proteins ubiquitinate histone H2B, leading to changes in chromatin dynamics and gene expression. Histone H2B ubiquitination occurs mainly at Lys-120 (Lys-123 for budding yeast), which promotes transcriptional activation by inhibiting chromatin compaction and promoting the formation of transcriptionally active chromatin [69,70]. RNF40 and its paralog RNF20 catalyse histone H2B monoubiquitination at Lys-120 by the E2 enzyme UbcH6, resulting in the formation of H2BK120Ub (also known as H2Bub1) and promoting HOX gene expression [44]. The RING finger E3 ubiquitin ligase MDM2 also possesses histone H2BK120 ubiquitination activity. MDM2 directly monoubiquitinates histone H2B by interacting with p53 [6], exerting transcriptional repression via its RING domain [46]. Furthermore, acting as a RING finger protein, MSL2 can form a complex with MSL1 that mediates ubiquitination at Lys-34 of histone H2B, resulting in the transcriptional activation of HOXA9 and MEIS1 [47].

RING finger proteins mediate the ubiquitination of histones H3&H4

The ubiquitination of histone H3 and histone H4 by RING finger proteins impacts multiple cellular processes, such as transcriptional activation, gene stability maintenance, transcriptional inactivation, DNA replication and DNA damage repair. The E3 ubiquitin protein ligase NEDD4 mediates ubiquitination on histone H3 at the Lys-23/36/37 residues [5] depending on its homologous to the E6-AP C-terminus (HECT domain). Through its DCAF8 (DDB1 and CUL4-associated factor 8) substrate receptor, the RING finger protein CRL4 performs ubiquitination on histone H3K79 within hepatocytes, resulting in the inactivation of fetal liver genes [52]. UHRF1 exerts ubiquitin ligase activity via its RING domain and ubiquitinates histone H3 at Lys-23 [11] and Lys-18 [51] in mammalian cells.

RING finger proteins can also ubiquitinate histone H4. The complex comprising CUL4, DDB and ROC1 mediates ubiquitination on histone H3 and histone H4, resulting in the release of these proteins from nucleosomes, further impairing the interaction between histones and DNA, and allowing repair proteins to reach damaged foci [16]. Furthermore, monoubiquitination of histone H4 at Lys-31 inhibits Mg²⁺-dependent chromatin compaction, similar to the suppression of chromatin compaction by H2BK120Ub [71].

Thus, RING finger proteins play a significant role in the ubiquitination of core histones that regulate chromatin remodeling. Multifaceted processes of histone ubiquitination impact gene expression, DNA repair, chromatin organization and other cellular processes, emphasizing the importance of regulating histone ubiquitination to maintain proper cellular function.

RING finger proteins regulate ATP-dependent chromatin remodeling complexes

RING finger proteins are involved in regulating several chromatin-remodeling complexes, such as SWI/SNF and FACT.

The SWI–SNF complex facilitates the transcription of a specific group of genes that depend on RNF20 and H2BK120Ub at the transcriptional level [72]. SMARCAD1 is activated in this context. The proximal coupling of ubiquitin conjugation to ER degradation (CUE) domain of SMARCAD1 recognizes the monoubiquitination of histone H2AK127/129 [73], depending on its ATPase activity to not only cleave DNA breaks but also promote DNA repair with high fidelity [74]. Moreover, the newly identified RING finger protein UNK ubiquitinates the SWI/SNF protein BAF60b in the presence of Rac GTPase [75]. This interaction highlights how RING finger proteins can directly influence the activities of chromatin-remodeling complexes, ultimately reshaping chromatin structure.

In a chromatin-mediated transcription system, the monoubiquitination of histone H2B by RNF20/40 is essential for regulating the transcription elongation factor PAF and the histone chaperone FACT [9]. Histone H2B monoubiquitination enables FACT to stimulate transcriptional elongation [9]. Beyond its involvement in transcriptional regulation, RNF20 remodels chromatin structure to permit homologous recombination (HR) repair proteins to assess DNA for repair in eukaryotes by ubiquitinating histone H2B [10]. Notably, a key component of FACT, SUPT16H, directly binds to RNF20. This interaction further underscores the close relationship between RING finger proteins and chromatin-remodeling complexes, with SUPT16H contributing to chromatin remodeling specifically for HR repair [76].

Therefore, the complex interaction between RING finger proteins and chromatin-remodeling complexes has significant implications for regulating gene transcription, repairing DNA damage and shaping chromatin structure.

Crosstalk between RING finger protein-mediated histone ubiquitination&other histone PTMs

Histone PTMs may interact and lead to different outcomes. This section will discuss the crosstalk between histone ubiquitination mediated by RING finger proteins and other histone PTMs, such as histone methylation and histone acetylation (Figure 2,&Tables 2&3).

Figure 2. The crosstalk between RING finger protein-mediated histone ubiquitination and other histone post-translational modifications, such as histone methylation and histone acetylation.

Different K sites of histones can be chemically modified by adding different small molecules. Ub and the corresponding RING finger proteins are shown in green, Me and the corresponding methyltransferases in orange, and Ac and the corresponding acetyltransferases in blue. Sharp arrows indicate facilitation; flat arrows indicate inhibition.

Ac: Acetyl; K: Lysine; Me: Methyl; Ub: Ubiquitination.

Table 2. RING finger proteins mediate the crosstalk between histone ubiquitination and histone methylation; the relationship between the interactions is also shown.

RING finger proteins	Ubiquitination	Methylation	Process of crosstalk	Ref.
PRC1	H2AK119	H3K27	PRC1 mediated H2AK119Ub promotes H3K27me3	[77,78]
PHF7	H2A	H3K4	PHF7 ubiquitinates H2A through binding H3K4me3/me2	[40]
MSL2	H2BK34	H3K4/79	MSL2 mediates H2BK34Ub, stimulating H3K4/K79 methylation	[47]
CLRC	H3K14	H3K9	H3K14 ubiquitination promotes H3K9 methylation	[49]
UHRF1	H3K18/23	H3K9	Uhrf1-dependent H3K18/K23Ub promotes H3K9 methylation	[11,51]
CRL4	H3K79	H3K9	CRL4 targets histone H3K79 and promotes H3K9 methylation	[52]
Cul4A	H4K31	H3K4/79	H4K31Ub by Cul4A Is necessary for methylation of H3K4/K79	[55]
BBAP	H4K91	H4K20	BBAP-mediated H4K91Ub promotes histone H4K20m	[56]

Table 3. RING finger proteins that mediate the crosstalk between histone ubiquitination and histone acetylation; the relationship between the interactions is also listed.

RING finger proteins	Ubiquitination	Acetylation	Process of crosstalk	Ref.
RNF168	H2AK15	H2BK120	RNF168-mediated H2AK15Ub inhibits H2BK120Ac.	[79]
PHF6	H2BK120	H2BK12	PHF6 mediates H2BK120Ub via H2BK12Ac recognition.	[45]
RNF8	H2B	H4K16	RNF8-mediated histone ubiquitination induces H4K16Ac.	[80]
NEDD4	H3K23/36/37	H3K9	NEDD4 ubiquitinates H3K23/36/37 and promotes H3K9Ac.	[5]

Crosstalk between RING finger protein-mediated histone ubiquitination&histone methylation

The complex interaction between histone ubiquitination mediated by RING finger proteins and the process of histone methylation is a noteworthy and crucial aspect of chromatin regulation. These two distinct PTMs work together to affect chromatin structure and gene expression by orchestrating a complex molecular conversation. Histone ubiquitination, catalyzed by RING finger E3 ligases, involves attaching ubiquitin moieties covalently to specific Lys residues on histones. This modification can lead to various outcomes, such as changes in nucleosome stability, the recruitment of effector proteins and DNA repair processes. In parallel, histone methylation, governed by histone methyltransferases, introduces methyl groups to specific histone Lys or arginine residues. Histone methylation signifies chromatin regions for distinct functional roles, influencing gene transcription and other chromatin-associated activities. Interestingly, these two modifications are not isolated events but are intricately linked through various mechanisms (Figure 2&Table 2).

Polycomb group (PcG) proteins are a group of transcription repressors that regulate target genes through chromatin modification. PcG proteins can be grouped into two major core protein complexes, PRC1 and PRC2, based on their biochemical and functional properties. These complexes play a crucial role in gene silencing through epigenetic processes and are integral to the regulation of gene expression in eukaryotic organisms. PRC1 is a macromolecular assembly consisting of multiple protein subunits, each with distinct roles in initiating gene silencing. One of the primary roles of this enzyme is facilitating the catalysis of monoubiquitination on histone H2A at Lys-119 (H2AK119Ub) [17]. By introducing this ubiquitin mark, PRC1 reinforces chromatin compaction and hinders the access of transcriptional machinery to target genes. PRC2 enhances the gene-silencing capabilities of PRC1 through its catalytic function in the di- and trimethylation of histone H3 at lysine 27 (H3K27me2/3) [81,82]. It is well documented that H2AK119Ub promotes recruiting PRC2-mediated H3K27me3 [77,78], while PRC2-mediated H3K27me3 facilitates PRC1-mediated H2AK119Ub [81,82]. PHF7 is a recently discovered E3 ligase that can selectively ubiquitinate histone H2A by binding to H3K4me3/me2 before the transition from histones to protamine occurs [40].

RNF20/40-catalyzed H2BK120Ub facilitates the recruitment of the histone methyltransferase COMPASS to achieve H3K4me2 and H3K4me3 [50,83,84], which forms a network of crosstalk between different histone modifications that contributes to the regulation of gene expression. For instance, MSL2-mediated H2BK34Ub [51] and Cul4A-mediated H4K31Ub [74] promote histone H3K4 and histone H3K79 methylation during histone crosstalk. Notably, histone H3K79 is one of the histone core sites that can be methylated, and this modification is catalyzed by Dot1L [85]. Importantly, the catalytic activity of Dot1L is dependent on H2BK120Ub, providing a fascinating example of crosstalk between histone PTMs [86,87]. Recently, significant progress has been made in understanding the structural basis of crosstalk between H2BK120Ub and methylation by depicting the cryo-electron microscopy structure of Dot1L in nucleosomes containing H2BK120Ub in both its active and inactive states. These findings provide insight into the mechanisms underlying the enhancement of histone H3K79 methylation by H2BK120Ub [88–91]. Specifically, in the active state, Dot1L establishes specific interactions with the ubiquitin moiety linked to histone H2BK120 and histone H3K79 [88].

Moving beyond H2BK120Ub, UHRF1 mediates histone H3K18/23 ubiquitination due to its histone- and DNA-binding activities [11]. Through the SRA domain [92], UHRF1 engages DNMT1 at hemi-methylation sites [93,94], thereby maintaining DNA methylation [11,95]. In addition, UHRF1 has been identified as a novel H3K9m-specific binding protein that is responsible for the formation of heterochromatin in mammalian cells via its PHD and SRA domain [96]. BBAP bestows a single ubiquitin molecule onto Lys-91 of histone H4. This action elicits a safeguarding reaction against DNA-damaging substances while fostering the association between 53BP1 and methylated histone H4K20 [56]. Upon DNA damage, BARD1 interacts with Lys-9-dimethylated histone H3 (H3K9me2) via a serine-protein kinase ATM-dependent mechanism that is not affected by RNF168 [97].

Crosstalk between RING finger protein-mediated histone ubiquitination&histone acetylation

Acetylation is a crucial PTM that regulates both chromatin structure and function. The dynamic interplay between RING finger protein-mediated histone ubiquitination and the complicated process of histone acetylation is pivotal for chromatin modulation (Figure 2 & Table 3). This interplay between these histone modifications represents a vital regulatory mechanism for chromatin dynamics and gene expression.

RNF168 mediates the ubiquitination of histone H2AK15 and inhibits histone H2BK120 acetylation, which is mediated by TIP60 [79]. Coincidentally, TIP60 can also mediate the acetylation of histone H2AK15 (H2AK15Ac) in vivo, and in turn, H2AK15Ac negatively regulates histone H2AK15 ubiquitination [79]. This intricate interplay highlights the reciprocal relationship between histone ubiquitination and acetylation. Histone H3 takes center stage in another fascinating interaction. By catalyzing ubiquitination on Lys residues 23/36/37 of histone H3, NEDD4 facilitates the recruitment of histone acetyltransferase GCN5, which leads to the subsequent acetylation of histone H3K9 and the transcriptional activation of some genes, including IL-1α, IL-1β and GCLM [5]. Here, the interplay between the ubiquitination and acetylation of histone finely regulates gene expression, highlighting the complexity of chromatin regulation.

The TIP60–UBC13 complex ubiquitinates histone H2AX (depending on prior histone H2AX acetylation), releases histone H2AX from damaged chromatin and facilitates DDR [98]. The precise coordination between these modifications emphasizes their functional importance in DNA-repair processes. Moreover, PHF6 introduces another level of complexity in recognizing H2BK12Ac, which in turn facilitates its E3 ubiquitin ligase function [45]. The ubiquitination of histones controlled by RNF8 triggers histone H4K16 acetylation, potentially serving as the primary phase of nucleosome elimination [80]. The E3 ligase UHRF2 upholds the stability of the acetyltransferase TIP60, regulating the levels of H3K9Ac and H3K14Ac through its RING finger domain [99].

The crosstalk between histone ubiquitination and methylation or acetylation reflects a layer of complexity in the complex regulatory landscape of chromatin biology. The interaction between histone ubiquitination and other histone PTMs elicits a cascade activation or inhibitory effect through mutual interactions, promoting or weakening each other’s function, and ultimately affecting gene transcription. This crosstalk enables the integration of diverse signals and pathways, affording precise control over gene expression in response to alterations in cellular cues. Understanding the intricate mechanisms regulating chromatin dynamics and their impact on development, disease and cellular functions can provide valuable insights. Unfortunately, the exact regulatory mechanism of the crosstalk is still poorly understood, and more in-depth analysis and exploration are still needed.

Biological functions of RING finger proteins in regulating chromatin remodeling

RING finger protein-mediated histone ubiquitination regulates gene transcription

Chromatin is a complex of DNA and associated proteins, consisting mainly of histones, that condense the DNA into a highly organized structure. The degree of chromatin condensation is a key factor in determining whether a gene is available for transcription (gene activation) or not (gene repression). Under normal physiological conditions, histone ubiquitination contributes to the fine-tuned control of the efficient expression of genes required for normal cellular processes. Chromatin structure alterations play a crucial role in regulating gene expression at the molecular level. These alterations, including changes in nucleosome positioning, histone modifications, DNA methylation and chromatin looping, have direct impacts on the extent of chromatin condensation, which in turn leads to changes in gene expression. RING finger protein-mediated histone ubiquitination can alter the structure of chromatin. RING finger protein can promote either an open or closed chromatin structure, depending on ubiquitinating the specific Lys residue of ubiquitinated histones. Consequently, changes in chromatin structure can directly affect the accessibility of genes to transcriptional machinery.

H2AK119Ub serves as a specific marker for epigenetic transcriptional repression, and histone H2B ubiquitination is a marker for transcriptional activation [100,101]. Histone H2AK119 ubiquitination mediated by RING finger proteins, including RING2-containing complexes (PRC1, hPRC1L, BCOR), PCGF2-RING1B, and RING finger protein 2A-HUB, UBR1 and UBR2, is strongly associated with transcriptional repression. Mechanistically, RING2-containing complex-catalyzed histone H2AK119 ubiquitination inhibits the activated form of the RNA polymerase II preinitiation complex [79] by blocking an early and integral event in preinitiation complex formation [102,103]. Histone H2AK119 ubiquitination enhances higher order chromatin compaction by promoting histone H1 binding with nucleosomes, thus further repressing gene transcription [104]. Furthermore, ubiquitination of histone H2AK119 catalyzed by 2A-HUB represses transcription by preventing FACT recruitment to the promoter region, hindering the elongation of RNA polymerase II during transcription [66].

The interaction between RNF20–RNF40 and RNA PAF1 together with RNA polymerase II at chromatin facilitates transcriptional elongation [9]. In addition, FACT is directed to chromatin sites with H2BK120Ub modification, causing physical disruption of the histone H2A–H2B dimer, leading to the reconstruction of a more open state of chromatin, thereby facilitating the crossing of RNA polymerase II and promoting transcriptional activation [9].

Therefore, the regulation of gene transcription is facilitated by RING finger protein-mediated histone ubiquitination, which interferes with the recruitment of transcription factors and RNA polymerase II and subsequently modifies chromatin remodeling.

RING finger protein-mediated histone ubiquitination participates in DDR

DDR signaling pathways are activated when cells are disturbed by environmental and endogenous factors. The incidence of DNA double-strand breaks [105] results in genome instability [106]. RING finger proteins are involved in DDR by interacting with DNA repair and HR proteins, coordinating structural changes in chromatin.

The ubiquitination of histone H2AK119, histone H2AK13/15 and histone H2AK127/129 plays critical functions in DDR. RING2–BMI1 is responsible for ubiquitinating histone H2AK119 and histone H2AX, consequently promoting double-strand break repair [107,108]. Histone H2A/H2AXK13/15 ubiquitination catalyzed by RNF8 and RNF168 is an essential signal in DDR. In a physiological context, RNF168 alters chromatin architecture through ubiquitinating histone H2A [68]. However, when DNA damage occurs, RNF168 is mobilized to damaged DNA loci, increasing the abundance of ubiquitinated proteins and initiating the downstream signaling pathways [109]. RNF8 first polyubiquitinates histone H1 (K63-linked ubiquitin chain) to recruit RNF168, monoubiquitinates histone H2A/H2AXK13/15 and finally extends the K63-linked ubiquitin chain by RNF8 to activate repair factors [68,110]. Furthermore, BRCA1 is associated with BARD1 [52] and is located at the central region of the lesion with HP1α to catalyze histone H2AK127/129 monoubiquitination. Histone H2AK127/129 monoubiquitination by the CUE domain of SMARCAD1 [73] cleaves DNA breaks and promotes DNA repair with high fidelity depending on its ATPase activity [74].

H2BK120Ub is related to transcriptional activation and acts as a signal to actively recruit DNA damage-associated proteins [111]. Histone H2BK120 monoubiquitination regulated by RNF20–RNF40 contributes to mammalian DDR [10], which facilitates histone H3K79 dimethylation for recruiting BRCA1, 53BP1 and RAD51 to DNA end resection for strand invasion [112–114]. RNF20-dependent histone H2B ubiquitination is also required for HR [111,115]. Moreover, the crosstalk between histone H2BK120 ubiquitination by RNF20–RNF40 and histone H3 methylation contributes to cell cycle arrest in response to DNA damage [116]. CUL4–DDB–ROC1 abolishes the interaction between histones and DNA, ultimately facilitating the effective enlistment of repair proteins to areas of DNA damage [16].

In yeast, Rad5 as an ATP-dependent chromatin remodeler [117] possesses a RING finger domain that confers its E3 ubiquitin-protein ligase activity [118]. Rad5 can polyubiquitinate PCNA and plays a critical role in postreplication repair [119]. Two mammalian Rad5 homologs are SHPRH and HLTF [120]. SHPRH and HLTF mediate polyubiquitination of PCNA at Lys 164 via activating a pathway of error-free template switching during DNA replication [120–122]. SHPRH also participates in DNA repair and transcriptional regulation through chromatin ubiquitination [123,124].

Therefore, RING finger protein-mediated histone ubiquitination participates in DDR by providing a signal for DNA damage, recruiting repair factors to the damaged site and facilitating the repair process through chromatin remodeling. This involvement ensures the maintenance of genome integrity and prevents mutations and genomic instability, which may cause diseases such as cancer.

Development of targeted therapeutics for RING finger proteins

RING finger proteins are widely implicated in the onset and progression of multiple diseases, including cancer [8], and thus have the potential to become therapeutic targets. The PRC1 protein BMI1 can ubiquitinate histone H2AK119 and its high expression is correlated with shorter survival duration in acute myeloid leukemia cells [125]. A BMI1 inhibitor, unesbulin (also known as PTC596), has entered phase II clinical trials evaluating its effects against ovarian cancer (NCT03206645), leiomyosarcoma (NCT03761095), high-grade glioma and diffuse intrinsic pontine glioma (NCT03605550) [126].

A recent study has identified UHRF1 as a novel epigenetic target for malignant pleural mesothelioma (MPM) [127]. Studies have shown that mithramycin can reduce UHRF1 expression in vitro and in vivo, thus presenting a promising approach for targeting UHRF1 in MPM, although thus far no clinical trials evaluating UHRF1 have been conducted [127]. Interestingly, circUHRF1 is found to be present in an exosomal manner in the plasma of patients with hepatocellular carcinoma (HCC) [128]. HCC-derived exosomal circUHRF1 can induce natural killer (NK) cell exhaustion and decrease NK cell function and tumor infiltration, contributing to HCC resistance to anti-PD1 therapy [128]. Mechanistically, circUHRF1 inhibits NK cell-derived IFN-γ and TNF-α secretion and drives resistance toanti-PD1 immunotherapy in HCC patients [128]. Furthermore, circUHRF1 inhibits NK cell function by upregulating the expression of TIM-3 via the degradation of miR-449c-5p [128]. Thus, circUHRF1 is associated with immunosuppression by decreasing NK cell proportion and tumor infiltration, further guiding the resistance to anti-PD1 immunotherapy in HCC patients [128]. These emerging roles of UHRF1 and circUHRF1 in cancer, particularly in the context of MPM and HCC, offer exciting prospects for the development of novel therapeutic approaches [128]. The potential of mithramycin as a UHRF1 inhibitor in MPM is a promising area for further investigation.

The E3 ubiquitin ligase Rnf20 was identified as a potential negative regulator of Foxp3 via CRISPR screen, and therefore a suitable target for immunotherapies [129]. With the advancement of immunotherapy, there is an optimistic outlook for new therapeutic strategies, including those targeting UHRF1 and circUHRF1, which may ultimately improve the prognosis and treatment outcomes for cancer patients. However, continuing investigation and the initiation of clinical trials will be necessary to confirm the practical application of these potential therapies beyond preclinical findings.

Thus, it is reasonable to surmise that numerous RING proteins have the potential to serve as effective therapeutic targets for various diseases. Gaining insight into the functions of RING finger proteins and their associated complexes, including BRCA1–BARD1 [29,30], RNF20–RNF40 [9,44], KDM2B–PCGF1–RING1B [22,23] and CUL4–DDB–RBX1 [16], within chromatin intricacies will help to understand the underlying mechanisms driving cancers and other diseases. It will provide new perspectives that may lead to more precise and effective therapeutic approaches to combat various diseases.

Although a handful of small-molecule inhibitors that target RING finger proteins are currently undergoing clinical trials [122], no drugs targeting these proteins are currently available on the market. Consequently, there is still a substantial need to develop inhibitors that target RING proteins.

Conclusion

This review elucidates the roles and underlying mechanisms of RING finger proteins as E3 ubiquitin ligases in regulating chromatin remodeling by ubiquitinating various histone Lys sites and interacting with other histone PTMs, such as histone methylation and histone acetylation. RING finger protein-mediated histone ubiquitination is involved in regulating gene transcription and DNA damage response concerning numerous specific diseases, such as cancers and autoimmune diseases. Targeting these RING finger proteins and the complexes containing RING finger proteins might be potentially effective therapeutic strategies. Other potential alternatives for targeting RING finger proteins may also help improve therapeutic strategies, such as targeting upstream regulatory proteins of RING finger proteins. Finally, designing inhibitors to target the crosstalk between histone ubiquitination and histone methylation/acetylation may provide research directions for the subsequent development of therapeutic strategies for malignancy.

Future perspective

The potential clinical implications of targeting histone ubiquitination mediated by RING finger proteins are particularly exciting. By selectively modulating specific events of histone ubiquitination, it may be possible to manipulate gene-expression patterns associated with various diseases, including cancers. These processes can potentially open avenues for the development of innovative therapeutic interventions that can restore aberrant gene-expression profiles to a healthier state. Exploring the genetic alterations of RING finger proteins in cancer and other diseases will establish new avenues for future research and provide ideas for discovering new therapeutic strategies. Understanding the intricate crosstalk between histone ubiquitination and other epigenetic modifications, such as methylation and acetylation, has the potential to facilitate combinational therapies that harness synergistic effects. With the advancement of technology allowing for more precise manipulation of histone modifications, there is considerable potential for therapeutic advancements in this area.

Executive summary

Chromatin remodeling

• Chromatin remodeling involves switching between a relaxed state (euchromatin) and a more condensed state (heterochromatin).

• The loose arrangement of euchromatin facilitates transcription factor binding, leading to increased gene expression. Conversely, the tightly packed heterochromatin limits DNA accessibility, resulting in gene repression.

Post-translational modifications on histones

• Histone post-translational modifications (PTMs) mainly include histone methylation, acetylation, ubiquitination and phosphorylation.

• PTMs on histones serve as dynamic switches, influencing chromatin compaction and gene accessibility.

RING finger proteins&histone ubiquitination

• Histone ubiquitination primarily occurs at core histones and affects gene transcriptional regulation, DNA damage response (DDR) and the maintenance of chromatin integrity.

• Histone H2A ubiquitination mainly occurs at lysine (Lys) residues 119/127/129/13/15, contributing to gene repression and DDR.

• Histone H2B ubiquitination mainly occurs at Lys-120/34, which promotes transcriptional activation by inhibiting chromatin compaction.

• Histone H3 ubiquitination mainly occurs at Lys-14/18/23/36/37/79 and is related to transcriptional regulation, gene stability maintenance, DNA replication and DDR.

• Histone H4 ubiquitination mainly occurs at Lys-31/91, facilitating DDR.

RING finger proteins&ATP-dependent chromatin remodeling complexes

• RING finger proteins can modulate chromatin structure by ubiquitinating several chromatin-remodeling complexes, including SWI–SNF and FACT.

• The intricate interplay between RING finger proteins and chromatin-remodeling complexes has significant implications for regulating gene transcription, DNA damage response and chromatin structure.

Crosstalk between histone ubiquitination&other histone PTMs

• The crosstalk between histone ubiquitination and histone methylation is related to gene transcriptional regulation and DDR.

• The crosstalk between histone ubiquitination and methylation or acetylation enables the integration of diverse signals and pathways, affording precise control over gene expression in response to alterations in cellular cues.

Biological functions of RING finger proteins in regulating chromatin remodeling

• Histone H2AK119 ubiquitination is strongly associated with transcriptional repression, while histone H2B ubiquitination promotes gene transcription.

• The ubiquitination of histone H2A cleaves DNA breaks and promotes DNA repair.

• Histone H2BK120 monoubiquitination orchestrated by RNF20–RNF40 contributes to the mammalian DDR.

• Histone H3 and histone H4 ubiquitination facilitate the recruitment of DDR proteins.

Development of targeted therapeutics for RING finger proteins

• BMI1 inhibitor unesbulin/PTC596 has entered phase II clinical trials involving several malignant tumors.

• Mithramycin can reduce UHRF1 expression in vitro and in vivo, providing a potential strategy for targeting UHRF1 in malignant pleural mesothelioma.

• The E3 ubiquitin ligase Rnf20 may work as a potential target for immunotherapy.

Conclusion

• RING finger proteins regulate chromatin remodeling via ubiquitinating different histone lysine sites and interacting with other histone PTMs.

• RING finger protein-induced histone ubiquitination participates in gene transcriptional regulation and DDR related to some specific diseases, including cancers and autoimmune diseases.

• Designing inhibitors targeting these RING finger proteins or targeting upstream regulatory proteins of RING finger proteins would be an effective therapeutic strategy.

• Breaking the crosstalk between histone ubiquitination and other PTMs could be a good option for the development of therapeutic strategies.

Future perspective

• Gaining insights into the functions of RING finger proteins and related complexes containing RING finger protein within chromatin intricacies helps to understand the underlying mechanisms driving cancer and other disorders.

• Understanding the intricate crosstalk between histone ubiquitination and other epigenetic modifications, such as methylation and acetylation, could pave the way for combinational therapeutic approaches that harness synergistic effects.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

Medical writing support was provided by LetPub.

Financial disclosure

This study was supported by the HMRF grant (no. 20190152), the Guangdong-Hong Kong Technology Cooperation Funding Scheme (TCFS; no. GHX/092/21SZ) and RGC (no. 14115019). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.