scholarly journals Transcription factor-like 5 is a potential DNA/RNA-binding protein essential for maintaining male fertility in mice

2021 ◽  
Author(s):  
Weiya Xu ◽  
Yiyun Zhang ◽  
Dongdong Qin ◽  
Yiqian Gui ◽  
Shu Wang ◽  
...  

Transcription factor-like 5 (TCFL5) is a testis-specific protein that contains the basic helix-loop-helix domain, but the in vivo functions of TCFL5 remain unknown. Herein, we generated CRISPR/Cas9-mediated knockout mice to dissect the function of TCFL5 in mouse testes. Surprisingly, we found that it was difficult to generate homozygous mice with the Tcfl5 deletion since the heterozygous males (Tcfl5+/-) were infertile. We did; however, observe markedly abnormal phenotypes of spermatids and spermatozoa in the testes and epididymides of Tcfl5+/- mice. Mechanistically, we demonstrated that TCFL5 transcriptionally and post-transcriptionally regulated a set of genes participating in male germ cell development via TCFL5 ChIP-DNA and eCLIP-RNA high-throughput sequencing. We also identified a known RBP, FXR1 as an interacting partner of TCFL5 that may coordinate the transition and localization of TCFL5 in the nucleus. Collectively, we herein report for the first time that Tcfl5 is haploinsufficient in vivo and acts as a dual-function protein that mediates DNA and RNA to regulate spermatogenesis.

2021 ◽  
Author(s):  
Weiya Xu ◽  
Yiyun Zhang ◽  
Dongdong Qin ◽  
Yiqian Gui ◽  
Shu Wang ◽  
...  

Tissue-specific transcription factors often play key roles in the development of specific cell lineages. Transcription factor-like 5 (TCFL5) is a testis-specific protein that contains the basic helix-loop-helix domain, although the in vivo functions of TCFL5 remain unknown. Herein, we generated CRISPR/Cas9-mediated knockout mice to dissect the function of TCFL5 in mouse testes. Surprisingly, we found that it was difficult to generate homozygous mice with the Tcfl5 deletion since the heterozygous males (Tcfl5+/-) were infertile. We did, however, observe markedly abnormal phenotypes of spermatids and spermatozoa in the testes and epididymides of Tcfl5+/- mice. Mechanistically, we demonstrated that TCFL5 transcriptionally regulated a set of genes participating in male germ cell development, which we uncovered via RNA-sequencing and TCFL5 ChIP-sequencing. We also found that TCFL5 interacted with RNA-binding proteins (RBPs) that regulated RNA processing, and further identified the fragile X mental retardation gene 1, autosomal homolog (FXR1, a known RBP) as an interacting partner of TCFL5 that may coordinate the transition and localization of TCFL5 in the nucleus. Collectively, we herein report for the first time that Tcfl5 is haploinsufficient in vivo and hypothesize that TCFL5 may be a dual-function protein that mediates DNA and RNA to regulate spermatogenesis.


2021 ◽  
Author(s):  
Roxie C. Girardin ◽  
Janice Pata ◽  
Xiaohong Qin ◽  
Haixin Sui ◽  
Kathleen A. McDonough

ABSTRACTThe bacterium Mycobacterium tuberculosis (Mtb) must adapt to myriad host-associated stressors. A recently identified transcription factor, AbmR (ATP-binding mcr11-regulator), regulates expression of an essential stress-responsive small RNA (Mcr11) and inhibits the growth of Mtb. Previously, AbmR was found to make 39S complexes of unknown function. Here we report that AbmR 39S complexes are comprised of AbmR and co-purifying RNAs and that RNA-binding inhibits AbmR’s DNA-binding function. While AbmR binds DNA and regulates gene expression in a sequence specific manner, RNA-binding is not sequence specific. Amino acid R146 is important for DNA-binding but completely dispensable for RNA-binding and 39S complex formation, establishing that the RNA- and DNA-binding functions of AbmR are distinct. RNA bound by AbmR was protected from RNase digestion, supporting an RNA modulatory function for the 39S complex. We also found that abmR is required for optimal survival during treatment with the ATP-depleting antibiotic bedaquiline, which is associated with extended RNA stability. These data establish a paradigm wherein a transcription factor assembles into large complexes to transition between mutually exclusive DNA-binding gene regulatory and RNA-binding RNA modulatory functions. Our findings indicate that AbmR is a dual-function protein that may have novel RNA regulatory roles in stress adapted Mtb.


Development ◽  
1997 ◽  
Vol 124 (17) ◽  
pp. 3263-3272 ◽  
Author(s):  
T. Roztocil ◽  
L. Matter-Sadzinski ◽  
C. Alliod ◽  
M. Ballivet ◽  
J.M. Matter

Genes encoding transcription factors of the helix-loop-helix family are essential for the development of the nervous system in Drosophila and vertebrates. Screens of an embryonic chick neural cDNA library have yielded NeuroM, a novel neural-specific helix-loop-helix transcription factor related to the Drosophila proneural gene atonal. The NeuroM protein most closely resembles the vertebrate NeuroD and Nex1/MATH2 factors, and is capable of transactivating an E-box promoter in vivo. In situ hybridization studies have been conducted, in conjunction with pulse-labeling of S-phase nuclei, to compare NeuroM to NeuroD expression in the developing nervous system. In spinal cord and optic tectum, NeuroM expression precedes that of NeuroD. It is transient and restricted to cells lining the ventricular zone that have ceased proliferating but have not yet begun to migrate into the outer layers. In retina, NeuroM is also transiently expressed in cells as they withdraw from the mitotic cycle, but persists in horizontal and bipolar neurons until full differentiation, assuming an expression pattern exactly complementary to NeuroD. In the peripheral nervous system, NeuroM expression closely follows cell proliferation, suggesting that it intervenes at a similar developmental juncture in all parts of the nervous system. We propose that availability of the NeuroM helix-loop-helix factor defines a new stage in neurogenesis, at the transition between undifferentiated, premigratory and differentiating, migratory neural precursors.


2020 ◽  
Vol 71 (20) ◽  
pp. 6311-6327
Author(s):  
Lincheng Zhang ◽  
Jing Kang ◽  
Qiaoli Xie ◽  
Jun Gong ◽  
Hui Shen ◽  
...  

Abstract Ethylene signaling pathways regulate several physiological alterations that occur during tomato fruit ripening, such as changes in colour and flavour. The mechanisms underlying the transcriptional regulation of genes in these pathways remain unclear, although the role of the MADS-box transcription factor RIN has been widely reported. Here, we describe a bHLH transcription factor, SlbHLH95, whose transcripts accumulated abundantly in breaker+4 and breaker+7 fruits compared with rin (ripening inhibitor) and Nr (never ripe) mutants. Moreover, the promoter activity of SlbHLH95 was regulated by RIN in vivo. Suppression of SlbHLH95 resulted in reduced sensitivity to ethylene, decreased accumulation of total carotenoids, and lowered glutathione content, and inhibited the expression of fruit ripening- and glutathione metabolism-related genes. Conversely, up-regulation of SlbHLH95 in wild-type tomato resulted in higher sensitivity to ethylene, increased accumulation of total carotenoids, slightly premature ripening, and elevated accumulation of glutathione, soluble sugar, and starch. Notably, overexpression of SlbHLH95 in rin led to the up-regulated expression of fruit ripening-related genes (FUL1, FUL2, SAUR69, ERF4, and CNR) and multiple glutathione metabolism-related genes (GSH1, GSH2, GSTF1, and GSTF5). These results clarified that SlbHLH95 participates in the regulation of fruit ripening and affects ethylene sensitivity and multiple metabolisms targeted by RIN in tomato.


1999 ◽  
Vol 19 (8) ◽  
pp. 5441-5452 ◽  
Author(s):  
Sarah J. Lee ◽  
Susan J. Baserga

ABSTRACT The function of the U3 small nucleolar ribonucleoprotein (snoRNP) is central to the events surrounding pre-rRNA processing, as evidenced by the severe defects in cleavage of pre-18S rRNA precursors observed upon depletion of the U3 RNA and its unique protein components. Although the precise function of each component remains unclear, since U3 snoRNA levels remain unchanged upon genetic depletion of these proteins, it is likely that the proteins themselves have significant roles in the cleavage reactions. Here we report the identification of two previously undescribed protein components of the U3 snoRNP, representing the first snoRNP components identified by using the two-hybrid methodology. By screening for proteins that physically associate with the U3 snoRNP-specific protein, Mpp10p, we have identified Imp3p (22 kDa) and Imp4p (34 kDa) (named for interacting with Mpp10p). The genes encoding both proteins are essential in yeast. Genetic depletion reveals that both proteins are critical for U3 snoRNP function in pre-18S rRNA processing at the A0, A1, and A2 sites in the pre-rRNA. Both Imp proteins associate with Mpp10p in vivo, and both are complexed only with the U3 snoRNA. Conservation of RNA binding domains between Imp3p and the S4 family of ribosomal proteins suggests that it might associate with RNA directly. However, as with other U3 snoRNP-specific proteins, neither Imp3p nor Imp4p is required for maintenance of U3 snoRNA integrity. Imp3p and Imp4p are therefore novel protein components specific to the U3 snoRNP with critical roles in pre-rRNA cleavage events.


2004 ◽  
Vol 24 (16) ◽  
pp. 7188-7196 ◽  
Author(s):  
Marianna Rodova ◽  
Kevin F. Kelly ◽  
Michael VanSaun ◽  
Juliet M. Daniel ◽  
Michael J. Werle

ABSTRACT Rapsyn is a synapse-specific protein that is required for clustering acetylcholine receptors at the neuromuscular junction. Analysis of the rapsyn promoter revealed a consensus site for the transcription factor Kaiso within a region that is mutated in a subset of patients with congenital myasthenic syndrome. Kaiso is a POZ-zinc finger family transcription factor which recognizes the specific core consensus sequence CTGCNA (where N is any nucleotide). Previously, the only known binding partner for Kaiso was the cell adhesion cofactor, p120 catenin. Here we show that δ-catenin, a brain-specific member of the p120 catenin subfamily, forms a complex with Kaiso. Antibodies against Kaiso and δ-catenin recognize proteins in the nuclei of C2C12 myocytes and at the postsynaptic domain of the mouse neuromuscular junction. Endogenous Kaiso in C2C12 cells coprecipitates with the rapsyn promoter in vivo as shown by chromatin immunoprecipitation assay. Minimal promoter assays demonstrated that the rapsyn promoter can be activated by Kaiso and δ-catenin; this activation is apparently muscle specific. These results provide the first experimental evidence that rapsyn is a direct sequence-specific target of Kaiso and δ-catenin. We propose a new model of synapse-specific transcription that involves the interaction of Kaiso, δ-catenin, and myogenic transcription factors at the neuromuscular junction.


1996 ◽  
Vol 151 (3) ◽  
pp. 491-499 ◽  
Author(s):  
Y Liu ◽  
P Cserjesi ◽  
A Nifuji ◽  
E N Olson ◽  
M Noda

Abstract Scleraxis is a recently identified transcription factor with a basic helix-loop-helix motif, which is expressed in sclerotome during embryonic development. We have examined the expression of scleraxis mRNA in rat osteoblastic cells and found that the scleraxis gene was expressed as a 1·2 kb mRNA species in osteoblastic osteosarcoma ROS 17/2·8 cells. The scleraxis mRNA expression was enhanced by type-β transforming growth factor (TGFβ) treatment. The TGFβ effect was observed in a dosedependent manner starting at 0·2 ng/ml and saturating at 2 ng/ml. The effect was time-dependent and was first observed within 12 h and peaked at 24 h. The TGFβ effect was blocked by cycloheximide, while no effect on scleraxis mRNA stability was observed. TGFβ treatment enhanced scleraxis-E box (Scx-E) binding activity in the nuclear extracts of ROS17/2·8 cells. Furthermore, TGFβ enhanced transcriptional activity of the CAT constructs which contain the Scx-E box sequence. TGFβ treatment also enhanced scleraxis gene expression in osteoblastenriched cells derived from primary rat calvaria. These findings indicated for the first time that the novel helixloop-helix type transcription factor (scleraxis) mRNA is expressed in osteoblasts and its expression is regulated by TGFβ. Journal of Endocrinology (1996) 151, 491–499


1996 ◽  
Vol 16 (4) ◽  
pp. 1714-1721 ◽  
Author(s):  
F Argenton ◽  
Y Arava ◽  
A Aronheim ◽  
M D Walker

The E2A protein is a mammalian transcription factor of the helix-loop-helix family which is implicated in cell-specific gene expression in several cell lineages. Mouse E2A contains two independent transcription activation domains, ADI and ADII; whereas ADI functions effectively in a variety of cultured cell lines, ADII shows preferential activity in pancreatic beta cells. To analyze this preferential activity in an in vivo setting, we adapted a system involving transient gene expression in microinjected zebra fish embryos. Fertilized one- to four-cell embryos were coinjected with an expression plasmid and a reporter plasmid. The expression plasmids used encode the yeast Gal4 DNA-binding domain (DBD) alone, or Gal4 DBD fused to ADI, ADII, or VP16. The reporter plasmid includes the luciferase gene linked to a promoter containing repeats of UASg, the Gal4-binding site. Embryo extracts prepared 24 h after injection showed significant luciferase activity in response to each of the three activation domains. To determine the cell types in which the activation domains were functioning, a reporter plasmid encoding beta-galactosidase and then in situ staining of whole embryos were used. Expression of ADI led to activation in all major groups of cell types of the embryo (skin, sclerotome, myotome, notochord, and nervous system). On the other hand, ADII led to negligible expression in the sclerotome, notochord, and nervous system and much more frequent expression in the myotome. Parallel experiments conducted with transfected mammalian cells have confirmed that ADII shows significant activity in myoblast cells but little or no activity in neuronal precursor cells, consistent with our observations in zebra fish. This transient-expression approach permits rapid in vivo analysis of the properties of transcription activation domains: the data show that ADII functions preferentially in cells of muscle lineage, consistent with the notion that certain activation domains contribute to selective gene activation in vivo.


2019 ◽  
Author(s):  
Andreas U. Müller ◽  
Marc Leibundgut ◽  
Nenad Ban ◽  
Eilika Weber-Ban

AbstractIn mycobacteria, transcriptional activator PafBC is responsible for upregulating the majority of genes induced by DNA damage. Understanding the mechanism of PafBC activation is impeded by a lack of structural information on this transcription factor that contains a widespread, but poorly understood WYL domain frequently encountered in bacterial transcription factors. Here, we determined the crystal structure ofArthrobacter aurescensPafBC. The protein consists of two modules, each harboring an N-terminal helix-turn-helix DNA binding domain followed by a central WYL and a C-terminal extension (WCX) domain. The WYL domains exhibit Sm-folds, while the WCX domains adopt ferredoxin-like folds, both characteristic for RNA binding proteins. Our results suggest a mechanism of regulation in which WYL domain-containing transcription factors may be activated by binding RNA molecules. Using anin vivomutational screen inMycobacterium smegmatis, we identify potential co-activator binding sites on PafBC.


1987 ◽  
Vol 7 (8) ◽  
pp. 2947-2955
Author(s):  
A Y Jong ◽  
M W Clark ◽  
M Gilbert ◽  
A Oehm ◽  
J L Campbell

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.


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