BRDT, a member of the BET family of double bromodomain‐containing proteins, is essential for spermatogenesis in the mouse and has been postulated to be a key regulator of transcription in meiotic and post‐meiotic cells. To understand the function of BRDT in these processes, we first characterized the genome‐wide distribution of the BRDT binding sites, in particular within gene units, by ChIP‐Seq analysis of enriched fractions of pachytene spermatocytes and round spermatids. In both cell types, BRDT binding sites were mainly located in promoters, first exons, and introns of genes. BRDT binding sites in promoters overlapped with several histone modifications and histone variants associated with active transcription, and were enriched for consensus sequences for specific transcription factors, including MYB, RFX, ETS, and ELF1 in pachytene spermatocytes, and JunD, c‐Jun, CRE, and RFX in round spermatids. Subsequent integration of the ChIP‐seq data with available transcriptome data revealed that stage‐specific gene expression programs are associated with BRDT binding to their gene promoters, with most of the BDRT‐bound genes being upregulated. Gene Ontology analysis further identified unique sets of genes enriched in diverse biological processes essential for meiosis and spermiogenesis between the two cell types, suggesting distinct developmentally stage‐specific functions for BRDT. Taken together, our data suggest that BRDT cooperates with different transcription factors at distinctive chromatin regions within gene units to regulate diverse downstream target genes that function in male meiosis and spermiogenesis.
Structural basis of non-canonical transcriptional regulation by the σA-bound iron-sulfur protein WhiB1 in M. tuberculosis
Abstract WhiB1 is a monomeric iron–sulfur cluster-containing transcription factor in the WhiB-like family that is widely distributed in actinobacteria including the notoriously persistent pathogen Mycobacterium tuberculosis (M. tuberculosis). WhiB1 plays multiple roles in regulating cell growth and responding to nitric oxide stress in M. tuberculosis, but its underlying mechanism is unclear. Here we report a 1.85 Å-resolution crystal structure of the [4Fe–4S] cluster-bound (holo-) WhiB1 in complex with the C-terminal domain of the σ70-family primary sigma factor σA of M. tuberculosis containing the conserved region 4 (σA4). Region 4 of the σ70-family primary sigma factors is commonly used by transcription factors for gene activation, and holo-WhiB1 has been proposed to activate gene expression via binding to σA4. The complex structure, however, unexpectedly reveals that the interaction between WhiB1 and σA4 is dominated by hydrophobic residues in the [4Fe–4S] cluster binding pocket, distinct from previously characterized canonical σ704-bound transcription activators. Furthermore, we show that holo-WhiB1 represses transcription by interaction with σA4in vitro and that WhiB1 must interact with σA4 to perform its essential role in supporting cell growth in vivo. Together, these results demonstrate that holo-WhiB1 regulates gene expression by a non-canonical mechanism relative to well-characterized σA4-dependent transcription activators.
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- Award ID(s):
- 1846908
- NSF-PAR ID:
- 10174033
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 48
- Issue:
- 2
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- 501 to 516
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Telomerase reverse transcriptase (
TERT ) activation plays an important role in cancer development by enabling the immortalization of cells.TERT regulation is multifaceted, and its promoter methylation has been implicated in controlling expression through alteration in transcription factor binding. We have characterizedTERT promoter methylation, transcription factor binding, andTERT expression levels in five differentiated thyroid cancer (DTC) cell lines and six normal thyroid tissue samples by targeted bisulfite sequencing, ChIP‐qPCR, and qRT‐PCR. DTC cell lines express varying levels ofTERT and exhibitTERT promoter methylation patterns similar to patterns seen in other telomerase positive cancer cell lines. The minimal promoter immediately surrounding the transcription start site is hypomethylated, while further upstream portions show dense methylation. In contrast, theTERT promoter in normal thyroid tissue is largely unmethylated throughout and expressesTERT minimally. Transcription factor binding is also affected byTERT mutation status. The E‐twenty‐six (ETS) factor GABPA exhibitsTERT binding in theTERT mutant DTC cells only, and allele‐specific methylation patterns at the minimal promoter were observed as well, which may indicate allele‐specific factor recruitment at the minimal promoter. Furthermore, we identified binding sites for activators MYC and GSC in the hypermethylated upstream region, pointing to its possible importance inTERT regulation. Overall,TERT expression and telomerase activity depend on the interplay of multiple regulatory mechanisms includingTERT promoter methylation, mutation status, and recruitment of transcription factors. This work explores of the interplay between these regulatory mechanisms and offers insight into cellular control of active telomerase in human cancer. -
Henkin, Tina M. (Ed.)ABSTRACT Regulation of gene expression is critical for Mycobacterium tuberculosis to tolerate stressors encountered during infection and for nonpathogenic mycobacteria such as Mycobacterium smegmatis to survive environmental stressors. Unlike better-studied models, mycobacteria express ∼14% of their genes as leaderless transcripts. However, the impacts of leaderless transcript structures on mRNA half-life and translation efficiency in mycobacteria have not been directly tested. For leadered transcripts, the contributions of 5′ untranslated regions (UTRs) to mRNA half-life and translation efficiency are similarly unknown. In M. tuberculosis and M. smegmatis , the essential sigma factor, SigA, is encoded by a transcript with a relatively short half-life. We hypothesized that the long 5′ UTR of sigA causes this instability. To test this, we constructed fluorescence reporters and measured protein abundance, mRNA abundance, and mRNA half-life and calculated relative transcript production rates. The sigA 5′ UTR conferred an increased transcript production rate, shorter mRNA half-life, and decreased apparent translation rate compared to a synthetic 5′ UTR commonly used in mycobacterial expression plasmids. Leaderless transcripts appeared to be translated with similar efficiency as those with the sigA 5′ UTR but had lower predicted transcript production rates. A global comparison of M. tuberculosis mRNA and protein abundances failed to reveal systematic differences in protein/mRNA ratios for leadered and leaderless transcripts, suggesting that variability in translation efficiency is largely driven by factors other than leader status. Our data are also discussed in light of an alternative model that leads to different conclusions and suggests leaderless transcripts may indeed be translated less efficiently. IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis , is a major public health problem killing 1.5 million people globally each year. During infection, M. tuberculosis must alter its gene expression patterns to adapt to the stress conditions it encounters. Understanding how M. tuberculosis regulates gene expression may provide clues for ways to interfere with the bacterium’s survival. Gene expression encompasses transcription, mRNA degradation, and translation. Here, we used Mycobacterium smegmatis as a model organism to study how 5′ untranslated regions affect these three facets of gene expression in multiple ways. We furthermore provide insight into the expression of leaderless mRNAs, which lack 5′ untranslated regions and are unusually prevalent in mycobacteria.more » « less
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- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1093/nar/gkz1133
