TAF4b transcription networks regulating early oocyte differentiation.

Establishment of a healthy ovarian reserve is contingent upon numerous regulatory pathways during embryogenesis. Previously, mice lacking TBP-associated factor 4b (Taf4b) were shown to exhibit a diminished ovarian reserve. However, potential oocyte-intrinsic functions of TAF4b have not been examined. Here, we use a combination of gene expression profiling and chromatin mapping to characterize TAF4b-dependent gene regulatory networks in mouse oocytes. We find that Taf4b-deficient oocytes display inappropriate expression of meiotic, chromatin modification/organization, and X-linked genes. Furthermore, dysregulated genes in Taf4b-deficient oocytes exhibit an unexpected amount of overlap with dysregulated genes in oocytes from XO female mice, a mouse model of Turner Syndrome. Using Cleavage Under Targets and Release Using Nuclease (CUT&RUN), we observed TAF4b enrichment at genes involved in chromatin remodeling and DNA repair, some of which are differentially expressed in Taf4b-deficient oocytes. Interestingly, TAF4b target genes were enriched for Sp/Klf family and NFY target motifs rather than TATA-box motifs, suggesting an alternative mode of promoter interaction. Together, our data connect several gene regulatory nodes that contribute to the precise development of the mammalian ovarian reserve.

Evaluation of the effects of the T-type calcium channel enhancer SAK3 in a rat model of TAF1 deficiency.

The TATA-box binding protein associated factor 1 (TAF1) is part of the TFIID complex that plays a key role during the initiation of transcription. Variants of TAF1 are associated with neurodevelopmental disorders. Previously, we found that CRISPR/Cas9 based editing of the TAF1 gene disrupts the morphology of the cerebral cortex and blunts the expression as well as the function of the CaV3.1 (T-type) voltage gated calcium channel. Here, we tested the efficacy of SAK3 (ethyl 8'-methyl-2', 4-dioxo-2-(piperidin-1-yl)-2'H-spiro [cyclopentane-1, 3'-imidazo [1, 2-a] pyridine]-2-ene-3-carboxylate), a T-type calcium channel enhancer, in an animal model of TAF1 intellectual disability (ID) syndrome. At post-natal day 3, rat pups were subjected to intracerebroventricular (ICV) injection of either gRNA-control or gRNA-TAF1 CRISPR/Cas9 viruses. At post-natal day 21, the rat pups were given SAK3 (0.25 mg/kg, p.o.) or vehicle for 14 days (i.e. till post-natal day 35) and then subjected to behavioral, morphological, and molecular studies. Oral administration of SAK3 (0.25 mg/kg, p.o.) significantly rescued locomotion abnormalities associated with TAF1 gene editing. SAK3 treatment prevented the loss of cortical neurons and GFAP-positive astrocytes observed after TAF1 gene editing. In addition, SAK3 protected cells from apoptosis. SAK3 also restored the Brain-derived neurotrophic factor/protein kinase B/Glycogen Synthase Kinase 3 Beta (BDNF/AKT/GSK3ß) signaling axis in TAF1 edited animals. Finally, SAK3 normalized the levels of three GSK3ß substrates - CaV3.1, FOXP2, and CRMP2. We conclude that the T-type calcium channel enhancer SAK3 is beneficial against the deleterious effects of TAF1 gene-editing, in part, by stimulating the BDNF/AKT/GSK3ß signaling pathway.

Deletions of the Idh1, Eco1, Rom2, and Taf10 Genes Differently Control the Hyphal Growth, Drug Tolerance, and Virulence of Candida albicans.

The most recent genome-editing system called CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat system with associated protein 9-nuclease) was employed to delete four non-essential genes (i.e., Caeco1, Caidh1, Carom2, and Cataf10) individually to establish their gene functionality annotations in pathogen Candida albicans. The biological roles of these genes were investigated with respect to the cell wall integrity and biogenesis, calcium/calcineurin pathways, susceptibility of mutants towards temperature, drugs and salts. All the mutants showed increased vulnerability compared to the wild-type background strain towards the cell wall-perturbing agents, (antifungal) drugs and salts. All the mutants also exhibited repressed and defective hyphal growth and smaller colony size than control CA14. The cell cycle of all the mutants decreased enormously except for those with Carom2 deletion. The budding index and budding size also increased for all mutants with altered bud shape. The disposition of the mutants towards cell wall-perturbing enzymes disclosed lower survival and more rapid cell wall lysis events than in wild types. The pathogenicity and virulence of the mutants was checked by adhesion assay, and strains lacking rom2 and eco1 were found to possess the least adhesion capacity, which is synonymous to their decreased pathogenicity and virulence.

The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex.

Understanding the molecular pathways disrupted in motor neuron diseases is urgently needed. Here, we employed CRISPR knockout (KO) to investigate the functions of four ALS-causative RNA/DNA binding proteins (FUS, EWSR1, TAF15 and MATR3) within the RNAP II/U1 snRNP machinery. We found that each of these structurally related proteins has distinct roles with FUS KO resulting in loss of U1 snRNP and the SMN complex, EWSR1 KO causing dissociation of the tRNA ligase complex, and TAF15 KO resulting in loss of transcription factors P-TEFb and TFIIF. However, all four ALS-causative proteins are required for association of the ASC-1 transcriptional co-activator complex with the RNAP II/U1 snRNP machinery. Remarkably, mutations in the ASC-1 complex are known to cause a severe form of Spinal Muscular Atrophy (SMA), and we show that an SMA-causative mutation in an ASC-1 component or an ALS-causative mutation in FUS disrupts association between the ASC-1 complex and the RNAP II/U1 snRNP machinery. We conclude that ALS and SMA are more intimately tied to one another than previously thought, being linked via the ASC-1 complex.

Transcription of Nearly All Yeast RNA Polymerase II-Transcribed Genes Is Dependent on Transcription Factor TFIID.

Previous studies suggested that expression of most yeast mRNAs is dominated by either transcription factor TFIID or SAGA. We re-examined the role of TFIID by rapid depletion of S. cerevisiae TFIID subunits and measurement of changes in nascent transcription. We find that transcription of nearly all mRNAs is strongly dependent on TFIID function. Degron-dependent depletion of Taf1, Taf2, Taf7, Taf11, and Taf13 showed similar transcription decreases for genes in the Taf1-depleted, Taf1-enriched, TATA-containing, and TATA-less gene classes. The magnitude of TFIID dependence varies with growth conditions, although this variation is similar genome-wide. Many studies have suggested differences in gene-regulatory mechanisms between TATA and TATA-less genes, and these differences have been attributed in part to differential dependence on SAGA or TFIID. Our work indicates that TFIID participates in expression of nearly all yeast mRNAs and that differences in regulation between these two gene categories is due to other properties.

A central role for TFIID in the pluripotent transcription circuitry.

Embryonic stem (ES) cells are pluripotent and characterized by open chromatin and high transcription levels, achieved through auto-regulatory and feed-forward transcription factor loops. ES-cell identity is maintained by a core of factors including Oct4 (also known as Pou5f1), Sox2, Klf4, c-Myc (OSKM) and Nanog, and forced expression of the OSKM factors can reprogram somatic cells into induced pluripotent stem cells (iPSCs) resembling ES cells. These gene-specific factors for RNA-polymerase-II-mediated transcription recruit transcriptional cofactors and chromatin regulators that control access to and activity of the basal transcription machinery on gene promoters. How the basal transcription machinery is involved in setting and maintaining the pluripotent state is unclear. Here we show that knockdown of the transcription factor IID (TFIID) complex affects the pluripotent circuitry in mouse ES cells and inhibits reprogramming of fibroblasts. TFIID subunits and the OSKM factors form a feed-forward loop to induce and maintain a stable transcription state. Notably, transient expression of TFIID subunits greatly enhanced reprogramming. These results show that TFIID is critical for transcription-factor-mediated reprogramming. We anticipate that, by creating plasticity in gene expression programs, transcription complexes such as TFIID assist reprogramming into different cellular states.

Dual functions of TAF7L in adipocyte differentiation.

The diverse transcriptional mechanisms governing cellular differentiation and development of mammalian tissue remains poorly understood. Here we report that TAF7L, a paralogue of TFIID subunit TAF7, is enriched in adipocytes and white fat tissue (WAT) in mouse. Depletion of TAF7L reduced adipocyte-specific gene expression, compromised adipocyte differentiation, and WAT development as well. Ectopic expression of TAF7L in myoblasts reprograms these muscle precursors into adipocytes upon induction. Genome-wide mRNA-seq expression profiling and ChIP-seq binding studies confirmed that TAF7L is required for activating adipocyte-specific genes via a dual mechanism wherein it interacts with PPARγ at enhancers and TBP/Pol II at core promoters. In vitro binding studies confirmed that TAF7L forms complexes with both TBP and PPARγ. These findings suggest that TAF7L plays an integral role in adipocyte gene expression by targeting enhancers as a cofactor for PPARγ and promoters as a component of the core transcriptional machinery.DOI:http://dx.doi.org/10.7554/eLife.00170.001.

Specific defects in different transcription complexes compensate for the requirement of the negative cofactor 2 repressor in Saccharomyces cerevisiae.

Negative cofactor 2 (NC2) has been described as an essential and evolutionarily conserved transcriptional repressor, although in vitro and in vivo experiments suggest that it can function as both a positive and a negative effector of transcription. NC2 operates by interacting with the core promoter and components of the basal transcription machinery, like the TATA-binding protein (TBP). In this work, we have isolated mutants that suppress the growth defect caused by the depletion of NC2. We have identified mutations affecting components of three different complexes involved in the control of basal transcription: the mediator, TFIIH, and RNA pol II itself. Mutations in RNA pol II include both overexpression of truncated forms of the two largest subunits (Rpb1 and Rpb2) and reduced levels of these proteins. Suppression of NC2 depletion was also observed by reducing the amounts of the mediator essential components Nut2 and Med7, as well as by deleting any of the nonessential mediator components, except Med2, Med3, and Gal11 subunits. Interestingly, the Med2/Med3/Gal11 triad forms a submodule within the mediator tail. Our results support the existence of different components within the basic transcription complexes that antagonistically interact with the NC2 repressor and suggest that the correct balance between the activities of specific positive and negative components is essential for cell growth.

TTP and BRF proteins nucleate processing body formation to silence mRNAs with AU-rich elements.

In mammalian cells, mRNAs with AU-rich elements (AREs) are targeted for translational silencing and rapid degradation. Here we present evidence that in human cells the proteins Tristetraprolin (TTP) and BRF-1 deliver ARE-mRNAs to processing bodies (PBs), cytoplasmic assemblies of mRNAs, and associated factors that promote translational silencing and mRNA decay. First, depletion of endogenous TTP and BRF proteins, or overexpression of dominant-negative mutant TTP proteins, impairs the localization of reporter ARE-mRNAs in PBs. Second, TTP and BRF-1 localize tethered mRNAs to PBs. Third, TTP can nucleate PB formation on untranslated mRNAs even when other mRNAs are trapped in polysomes by cycloheximide treatment. ARE-mRNA localization in PBs is mediated by the TTP N- and C-terminal domains and occurs downstream from mRNA polysome release, which in itself is not sufficient for mRNA PB localization. The accumulation of ARE-mRNAs in PBs is strongly enhanced when the mRNA decay machinery is rendered limiting by mRNA decay enzyme depletion or TTP/BRF-1 overexpression. Based on these observations, we propose that the PB functions as a reservoir that sequesters ARE-mRNAs from polysomes, thereby silencing ARE-mRNA function even when mRNA decay is delayed. This function of the PB can likely be extended to other mRNA silencing pathways, such as those mediated by microRNAs, premature termination codons, and mRNA deadenylation.

Ovarian granulosa cell survival and proliferation requires the gonad-selective TFIID subunit TAF4b.

Oocyte development in the mammalian ovary requires productive interactions with somatic granulosa cells of the ovarian follicle. Proliferating granulosa cells support the progression of follicular growth and maturation, multiplying dramatically as it unfolds. The cell cycle recruitment of granulosa cells is regulated at least in part by hormones such as follicle-stimulating hormone (FSH) and estrogen. Follicles recruited into the growth phase following formation of multiple layers of granulosa cells have two major fates: either to continue proliferation followed by differentiation, or to die by programmed cell death, or atresia. While many of the signaling pathways orchestrating ovarian follicle development are known, the downstream transcriptional regulators that integrate such signals in the mammalian ovary remain to be defined. Recent experiments in diverse organisms have revealed multiple instances of gonad-selective components of the basal transcriptional machinery. One such protein, TAF4b, is a gonadal-enriched coactivator subunit of the TFIID complex required for normal female fertility in the mouse. To determine the etiology of female infertility of the TAF4b-deficient mice, we have determined multiple functions of TAF4b during postnatal ovarian follicle development. Here we demonstrate that the TAF4b protein is expressed in the granulosa cell compartment of the mammalian ovarian follicle. Furthermore, TAF4b-deficient mouse ovaries contain reduced numbers of primordial as well as growing follicles and a concomitant increased proportion of apoptotic follicles in comparison to wild type counterparts. Importantly, TAF4b-null follicles are largely resistant to induction of proliferation in response to multiple hormonal stimuli including estrogen and FSH and demonstrate compromised granulosa cell survival. Together, these data suggest that TAF4b integrates a program of granulosa cell gene expression required for normal ovarian follicle survival and proliferation in response to diverse ovarian signaling events.

TAF10 is required for the establishment of skin barrier function in foetal, but not in adult mouse epidermis.

TFIID, composed of the TATA box binding protein (TBP) and 13 TBP-associated factors (TAFs), plays a role in nucleating the assembly of the RNA polymerase II preinitiation complexes on protein coding genes. TAF10 (formerly TAF(II)30) is shared between TFIID and other transcription regulatory complexes (i.e. SAGA, TFTC, STAGA and PCAF/GCN5). TAF10 is an essential transcription factor during very early stages of mouse embryo development. To study the in vivo function of TAF10 in cellular differentiation and proliferation at later stages, the role of TAF10 was analysed in keratinocytes during skin development and adult epidermal homeostasis. We demonstrate that ablation of TAF10 in keratinocytes of the forming epidermis affects the expression of some, but not all genes, impairs keratinocyte terminal differentiation and alters skin permeability barrier functions. In contrast, loss of TAF10 in keratinocytes of adult epidermis did not (i) modify the expression of tested genes, (ii) affect epidermal homeostasis and (iii) impair acute response to UV irradiation or skin regeneration after wounding. Thus, this study demonstrates for the first time a differential in vivo requirement for a mammalian TAF for the regulation of gene expression depending on the cellular environment and developmental stage of the cell.

Maintenance of spermatogenesis requires TAF4b, a gonad-specific subunit of TFIID.

The establishment and maintenance of spermatogenesis in mammals requires specialized networks of gene expression programs in the testis. The gonad-specific TAF4b component of TFIID (formerly TAF(II)105) is a transcriptional regulator enriched in the mouse testis. Herein we show that TAF4b is required for maintenance of spermatogenesis in the mouse. While young Taf4b-null males are initially fertile, Taf4b-null males become infertile by 3 mo of age and eventually exhibit seminiferous tubules devoid of germ cells. At birth, testes of Taf4b-null males appear histologically normal; however, at post-natal day 3 gonocyte proliferation is impaired and expression of spermatogonial stem cell markers c-Ret, Plzf, and Stra8 is reduced. Together, these data indicate that TAF4b is required for the precise expression of gene products essential for germ cell proliferation and suggest that TAF4b may be required for the regulation of spermatogonial stem cell specification and proliferation that is obligatory for normal spermatogenic maintenance in the adult.