COL1A1 proximal promoter topology regulates its transcriptional response to transforming growth factor β

ABSTRACT

Objective

The COL1A1 proximal promoter contains two GC-rich regions and two inverted CCAAT boxes. The transcription factors Sp1 and CBF bind to the GC sequence at −122 to −115 bp and the inverted CCAAT box at −101 to −96 bp, respectively, and stimulate COL1A1 transcriptional activity.

Methods

To further define the regulatory mechanisms controlling COL1A1 expression by Sp1 and CBF, we introduced 2, 4, 6, or 8 thymidine nucleotides (T-tracts) at position −111 bp of the COL1A1 gene promoter to increase the physical distance between these two binding sites and examined in vitro the transcriptional activities of the resulting constructs and their response to TGF-β1.`

Results

Insertion of 2 or 4 nucleotides decreased COL1A1 promoter activity by up to 70%. Furthermore, the expected increase in COL1A1 transcription in response to TGF-β1 was abolished. Computer modeling of the modified DNA structure indicated that increasing the physical distance between the Sp1 and CBF binding sites introduces a rotational change in the DNA topology that disrupts the alignment of Sp1 and CBF binding sites and likely alters protein–protein interactions among these transcription factors or their associated co-activators.

Conclusion

The topology of the COL1A1 proximal promoter is crucial in determining the transcriptional activity of the gene and its response to the stimulatory effects of TGF-β1.

KEYWORDS:

• Systemic sclerosis
• TGF-β1
• COL1A1 promoter
• Sp1
• CBF

Acknowledgments

The expert assistance of Alana Pagano in the preparation of the manuscript is gratefully acknowledged.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Authors contributions

• Study design and conception: SAJ and EH.

• Performance of experiments: EH and SG.

• Analysis and interpretation of results: EH, SP-V, and SAJ.

• Writing of manuscript: SP-V and SAJ.

Institutional review board statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Thomas Jefferson University (Protocol #06F.186, approved on 2/11/2022).

Informed consent statement

Informed consent was obtained from all subjects involved in this study.

Additional information

Funding

Supported by NIH Grant RO-1 AR519106 to SAJ.