Formin-mediated epigenetic maintenance of centromere identity

ABSTRACT

Accurate chromosome segregation in mammalian cells is guided by the centromere, a specialized chromosome region defined by the histone H3 variant centromere protein A (CENP-A). It is not well understood how cells maintain CENP-A levels at centromeres while continuously going through genome replications and cell divisions. A MgcRacGAP-dependent small GTPase molecular switch has been shown as essential for centromeric CENP-A maintenance. By using quantitative imaging, pulse-chase and live cell analysis, a recent work has suggested that the diaphanous formin mDia2, a well-established small GTPase effector, functions downstream of this small GTPase pathway to maintain CENP-A levels at centromeres. A constitutively active mDia2 construct is able to rescue the CENP-A loading defect caused by MgcRacGAP depletion. This study has uncovered an unsuspected role of the cytoskeleton protein mDia2 as an effector of the MgcRacGAP-dependent small GTPase signaling inside the nucleus to participate in the epigenetic regulation of centromere maintenance during cell cycle.

KEYWORDS:

• cell cycle
• centromere
• CENP-A
• cytoskeleton
• diaphanous formin
• epigenetics
• MgcRacGAP
• mDia2

Faithful transmission of genetic materials during cell divisions requires 2 sets of replicated chromosomes being accurately segregated into 2 daughter cells. In mitosis, chromosome segregation is driven by the mitotic spindle, where microtubule polymers form mechanical linkages to the kinetochore—a proteinaceous structure assembled at a specialized chromosomal region called the centromere.¹ To ensure accurate chromosome segregation, it is important that the presence and the position of the centromere on each chromosome are stably maintained over many rounds of cell divisions. Through the course of evolution, many eukaryotes today use a conserved protein called centromere protein A (CENP-A) to mark the identity of centromeric chromatin. This is an epigenetic mark, as CENP-A is a histone H3 variant that forms nucleosomes composing centromeric chromatin regardless of the DNA sequences involved.¹ When DNA replicates in S-phase, however, the number of preexisting marker molecules per centromere will be diluted by half as they are redistributed to 2 daughter chromatin strands. Therefore, an outstanding question in the field is how CENP-A, as the marker molecule, maintains its level at centromeres. We now know that mammalian cells address this issue by replenishing the CENP-A level per centromere in early G1 phase of the cell cycle, right after the previous round of genome division and before the next round of DNA replication.² Although several important stages and molecular players have been identified to license, load and stabilize newly synthesized CENP-A proteins at centromeres labeled with preexisted and diluted CENP-A,^3-7 a complete understanding is missing regarding how new CENP-A proteins become stably incorporated into centromeric nucleosomes during early G1.

A Rho family small GTPase molecular switch is required for new CENP-A incorporation at centromeres

Rho family small GTPase proteins along with their cognate guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) are generally distributed in the cytosol and participate in signaling regulation in membrane and cytoskeleton dynamics.^8,9 Recent studies have suggested nuclear functions of the Rho family small GTPase signaling. Net1, a RhoA specific GEF, and active RhoA have been found inside the nucleus.¹⁰ A nuclear pool of RhoA is specifically activated in a Net1-dependent manner upon ionizing radiation (IR).¹⁰ Loss of Ect2 and Net1 has been shown to suppress RhoB activation upon IR-induced DNA damage.¹¹ Rac1, another Rho family small GTPase protein, has a functional nuclear localization signal (NLS) for nuclear import mediated by karyopherin α2 and associates with numerous nuclear proteins.¹²

Leptomycin B (LMB) treatment results in a nuclear accumulation of the GTPase activating protein MgcRacGAP concurrent with a decrease in the cytoplasm.¹³ The guanine exchange factor Ect2 contains a NLS and is preferentially localized to the nucleus at steady state.¹⁴ Knocking down either MgcRacGAP or Ect2 for one cell cycle reduces the total CENP-A levels at the centromere by half, which is consistent with a defect in CENP-A replenishment.¹⁵ Furthermore, depleting Cdc42 or Rac1, but not RhoA, results in a similar phenotype as MgcRacGAP or ECT2 depletion, indicating that Cdc42 and/or Rac1 are the small GTPases involved in new CENP-A loading/maintenance.¹⁵ Thus, it is proposed that a molecular switch of Rho family small GTPase is essential in regulating epigenetic centromere maintenance by stabilizing newly loaded CENP-A.¹⁵ However, it is difficult to know the exact functional role for the small GTPase pathway in this process without identifying the downstream effector(s).

The formin mDia2 functions downstream of the MgcRacGAP-dependent GTPase pathway to maintain CENP-A levels at the centromere

The diaphanous formin family proteins function as effectors of the small GTPase signaling in diverse aspects of the cell cycle and are well-established cytoskeleton regulators involved in both actin and microtubule dynamics.^16,17 Three members of the diaphanous formin family, mDia1-3, have conserved domain structures and a similar mode of action but differed choices of upstream GTPase signaling pathways^18-27 (Fig. 1). While investigating kinetochore functions of formin mDia3^28,29 using quantitative imaging, we have unexpectedly observed that depleting mDia2, but not mDia3, results in an almost 50% reduction of total CENP-A levels at centromeres after one round of cell cycle.³⁰ Furthermore, ratiometric live cell imaging studies (Fig. 2) along with pulse chase approaches demonstrate that cells depleted of mDia2 are unable to maintain the continuous loading of new CENP-A onto centromeres.³⁰ The formin mDia2 have been shown to be the effector of either Rac or Cdc42,^20,21,23 2 small GTPases whose depletion results in a similar centromeric CENP-A reduction phenotype.¹⁵ Unlike mDia1 or mDia3, mDia2 shuttles from cytoplasm to nucleoplasm evidenced by nuclear accumulation upon LMB treatment³¹ and a clear nuclear distribution in early G1 cells (Fig. 1B).

Figure 1. Diaphanous formins are effectors of the small GTPase signaling. (A) Schematic diagram of the general structure and the mode of action of diaphanous formin proteins. GBD – GTPase binding domain; DID – Dia interacting domain; FH – Formin homology domain; DAD – Dia auto-inhibition domain. Small GTPase binding releases the auto-inhibitory interaction between the DID and the DAD domains. The FH1FH2 alone can be used as a constitutively active construct that functions without the GTPase signaling. (B) Comparison between the 3 members of diaphanous subfamily formins, with emphasis on the cognate upstream small GTPase signaling and the subcellular localization. Scale bar, 10 µm.

Figure 2. High resolution quantitative live-cell imaging for systematically measurement of total CENP-A levels at centromeres in real time. Left: overlaid plots showing ratiometric measurement of YFP-CENP-A levels from centromeres in multiple cells (light blue/red circles are raw data points, and dark blue/red lines are sample averages). Right: 2 representative frames of cells with YFP-CENP-A on the centromeres pseudocolored based on intensity levels. (Data were replotted from Liu and Mao., JCB, 2016³⁰ Scale bar, 10 µm.).

Diaphanous formins assume an auto-inhibited state resulted from an intra-molecular interaction between the DID and DAD domains. Upon small Rho GTPase binding at the N-terminus, the auto-inhibition of mDia formins is released to expose the formin homology domain (FH2) responsible for actin polymerization and microtubule stabilization.³² A short-version of mDia proteins consisting of formin homology domains without regulatory regions required for auto-inhibition (Fig. 1A) is constitutively active even in the absence of small GTPase binding.²² Such a constitutively active construct of mDia2 is able to rescue the CENP-A loading defect resulted from depletion of MgcRacGAP.³⁰ This epistatic analysis suggests that mDia2 is the effector downstream of the MgcRacGAP-mediated small GTPase switch in maintaining CENP-A levels at centromeres.

The identification of mDia2 as a small GTPase effector required for maintaining newly loaded CENP-A in early G1 has led to an obvious question: what exactly does mDia2 do in maintaining CENP-A levels at centromeres (Fig. 3)? Diaphanous formins are well known for their functions in polymerizing linear actin filaments and stabilizing microtubules.^33-35 The microtubule stabilization activity seems unlikely to be involved in mDia2's CENP-A function, as tubulins and microtubules are not normally found inside the nucleus.³⁶ On the contrary, recent works from multiple groups have implicated functional roles for the actin nucleation activity of formin proteins inside the nucleus.^36-38 As a potent actin nucleator, formin proteins promote actin polymerization inside the nucleus to participate in the regulation of serum response factor transcriptional activity³⁶ and the regulation of DNA damage response.³⁸ Consistent with the hypothesis that the actin nucleation activity of mDia2 is important for new CENP-A loading, constitutively active mDia2 constructs with point mutations that have been shown to be defective in polymerizing actin filaments in vitro and in vivo,¹⁷ unlike the wild-type constitutively active construct, are not able to rescue centromeric CENP-A levels upon depleting endogenous mDia2.³⁰

Figure 3. Schematic summary showing mDia2 functions downstream of the MgcRacGAP-dependent GTPase pathway to maintain CENP-A levels at the centromere. Depending on the GEF (Ect2) and GAP (MgcRacGAP), the Rho family small GTPase cycles between active form and inactive form, which is important for CENP-A maintenance at the centromeres. Diaphanous formin mDia2 has an epistatic relationship downstream of the MgcRacGAP-dependent small GTPase molecular switch and promotes G1 CENP-A loading/maintenance.

A new model of epigenetic regulation of centromeric nucleosome assembly

The assembly of CENP-A nucleosomes underlying the centromeric chromatin includes several discernable steps. Upon anaphase onset, released from the CDK-based inhibition and promoted by the Polo kinase 1 (Plk1)-based stimulation, the Mis18 complex (consisting Mis18α, Mis18β and Mis18 binding protein 1) localizes to the centromere to license the process of new CENP-A recruitment.^7,39 The Mis18 complex is crucial for the recruitment of Holliday junction recognition protein (HJURP), a chaperon that binds to CENP-A in a pre-nucleosomal complex, to the centromere to execute its nucleosome assembly functions for new CENP-A.^5,6,40 Notably, the localization of HJURP at G1 centromeres appears to be transient, as HeLa cells yield a 2∼3 hr time window in early G1 in which HJURP can be detected on the centromere. Knocking down mDia2 does not affect HJURP recruitment onto the centromere.³⁰ However, upon mDia2 depletion, the percentage of cells with their centromeres decorated with HJURP is substantially increased.³⁰ A reasonable explanation for a prolonged centromeric localization of HJURP is that the transient HJURP turnover at early G1 centromeres is delayed in the absence of mDia2. A stochastic model with numerical simulation suggests that extended dwelling time of HJURP molecules at centromeres can indeed cause not only significantly higher percentage of cells with HJURP positive centromeres but also a defective loading process over time, which is consistent with observations from live-cell imaging studies.³⁰ Therefore, in the absence of mDia2, HJURP-bound CENP-A cannot be stably incorporated into the centromeric nucleosomes (Fig. 3).

Concluding remarks

That the constitutively active form of mDia2 is able to rescue the reduced levels of CENP-A at centromeres caused by depletion of MgcRacGAP strongly suggests the formin mDia2 as the downstream effector of the small GTPase-mediated molecular switch in new CENP-A loading at centromeres. It is proposed that the small GTPase activity modifies newly incorporated CENP-A at the correct place as a late-G1 surveillance step by either adding or removing a mark (modification or conformation change).¹⁵ However, the identification of mDia2 as the downstream effector of this small GTPase pathway does not seem to support this hypothesis. First, ratiometric live-cell imaging analysis shows that depletion of mDia2 results in a failure in new CENP-A incorporation at centromeres in early G1.³⁰ And second, mDia2 depletion causes a prolonged centromeric localization of HJURP at G1,³⁰ which is consistent with a failed incorporation of CENP-A molecules into centromeric nucleosomes. Thus, one challenge for future studies will be to understand the exact functional roles for the small GTPase molecular switch and the formin mDia2 in regulating epigenetic centromere maintenance.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by a grant from the National Institute of Health (GM89768) to YM.