Regulation of Transcriptional Activators by DNA-Binding Domain Ubiquitination

Ubiquitin is a key component of the regulatory network that maintains gene expression in eukaryotes, yet the molecular mechanism(s) by which non-degradative ubiquitination modulates transcriptional activator (TA) function is unknown. Here endogenous p53, a stress-activated transcription factor required to maintain health, is stably monoubiquitinated, following pathway activation by IR or Nutlin-3 and localized to the nucleus where it becomes tightly associated with chromatin. Comparative structure–function analysis and in silico modelling demonstrate a direct role for DNA-binding domain (DBD) monoubiquitination in TA activation. When attached to the DBD of either p53, or a second TA IRF-1, ubiquitin is orientated towards, and makes contact with, the DNA. The contact is made between a predominantly cationic surface on ubiquitin and the anionic DNA. Our data demonstrate an unexpected role for ubiquitin in the mechanism of TA-activity enhancement and provides insight into a new level of transcriptional regulation

DSIF and RNA Polymerase II CTD Phosphorylation Coordinate the Recruitment of Rpd3S to Actively Transcribed Genes

Histone deacetylase Rpd3 is part of two distinct complexes: the large (Rpd3L) and small (Rpd3S) complexes. While Rpd3L targets specific promoters for gene repression, Rpd3S is recruited to ORFs to deacetylate histones in the wake of RNA polymerase II, to prevent cryptic initiation within genes. Methylation of histone H3 at lysine 36 by the Set2 methyltransferase is thought to mediate the recruitment of Rpd3S. Here, we confirm by ChIP–Chip that Rpd3S binds active ORFs. Surprisingly, however, Rpd3S is not recruited to all active genes, and its recruitment is Set2-independent. However, Rpd3S complexes recruited in the absence of H3K36 methylation appear to be inactive. Finally, we present evidence implicating the yeast DSIF complex (Spt4/5) and RNA polymerase II phosphorylation by Kin28 and Ctk1 in the recruitment of Rpd3S to active genes. Taken together, our data support a model where Set2-dependent histone H3 methylation is required for the activation of Rpd3S following its recruitment to the RNA polymerase II C-terminal domain

Genomic dissection of conserved transcriptional regulation in intestinal epithelial cells

Author summary The epithelium lining the intestine is an ancient animal tissue that serves as a primary site of nutrient absorption and interaction with microbiota. Its formation and function require complex patterns of gene transcription that vary along the intestine and in specialized intestinal epithelial cell (IEC) subtypes. However, it is unknown how the underlying transcriptional regulatory mechanisms have changed over the course of vertebrate evolution. Here, we used genome-wide profiling of mRNA levels and chromatin accessibility to identify conserved IEC genes and regulatory regions in 4 vertebrate species (zebrafish, stickleback, mouse, and human) separated from a common ancestor by 420 million years. We identified substantial similarities in genes expressed along the vertebrate intestine. These data disclosed putative conserved transcription factor binding sites (TFBS) enriched in accessible chromatin near IEC genes and in regulatory sites with accessibility restricted to IECs. Fluorescent reporter assays in transparent zebrafish showed that these regions, which frequently lacked sequence conservation, were still capable of driving conserved expression patterns. We also found a highly conserved region near mammalian and fish hes1 sufficient to drive expression in a specific population of IECs with active Notch signaling. These results establish a platform to define the conserved transcriptional networks underlying vertebrate IEC physiology

Conserved roles for Hnf4 family transcription factors in zebrafish development and intestinal function.

Transcription factors play important roles in the development of the intestinal epithelium and its ability to respond to endocrine, nutritional, and microbial signals. Hepatocyte nuclear factor 4 family nuclear receptors are liganded transcription factors that are critical for the development and function of multiple digestive organs in vertebrates, including the intestinal epithelium. Zebrafish have 3 hepatocyte nuclear factor 4 homologs, of which, hnf4a was previously shown to mediate intestinal responses to microbiota in zebrafish larvae. To discern the functions of other hepatocyte nuclear factor 4 family members in zebrafish development and intestinal function, we created and characterized mutations in hnf4g and hnf4b. We addressed the possibility of genetic redundancy amongst these factors by creating double and triple mutants which showed different rates of survival, including apparent early lethality in hnf4a; hnf4b double mutants and triple mutants. RNA sequencing performed on digestive tracts from single and double mutant larvae revealed extensive changes in intestinal gene expression in hnf4a mutants that were amplified in hnf4a; hnf4g mutants, but limited in hnf4g mutants. Changes in hnf4a and hnf4a; hnf4g mutants were reminiscent of those seen in mice including decreased expression of genes involved in intestinal function and increased expression of cell proliferation genes, and were validated using transgenic reporters and EdU labeling in the intestinal epithelium. Gnotobiotics combined with RNA sequencing also showed hnf4g has subtler roles than hnf4a in host responses to microbiota. Overall, phenotypic changes in hnf4a single mutants were strongly enhanced in hnf4a; hnf4g double mutants, suggesting a conserved partial genetic redundancy between hnf4a and hnf4g in the vertebrate intestine

RETINA-Specific Expression of Kcnv2 Is Controlled by Cone-Rod Homeobox (Crx) and Neural Retina Leucine Zipper (Nrl)

Cone dystrophy with supernormal rod response (CDSRR) is an autosomal recessive disorder that leads to progressive retinal degeneration with a distinct electroretinogram (ERG) phenotype. CDSRR patients show reduced sensitivity to dim light, augmented response to suprathreshold light and reduced response to flicker. The disorder is caused by mutations in the KCNV2 gene, which encodes the Kv11.1 subunit of a voltage-gated potassium channel. Here, we studied the retina-specific expression and cis-regulatory activity of the murine Kcnv2 gene using electroporation of explanted retinas. Using qRT-PCR profiling of early postnatal retinas, we showed that Kcnv2 expression increased towards P14, which marks the beginning of visual activity in mice. In vivo electroporation of GFP-Kcnv2 expressing plasmids revealed that Kv11.1 localizes to the inner segment membranes of adult P21 photoreceptors. Using bioinformatic prediction and chromatin immuno-precipitation (ChIP), we identified two Crx binding sites (CBS) and one Nrl binding site (NBS) in the Kcnv2 promoter. Reporter electroporation of the wild type promoter region induced strong DsRed expression, indicating high regulatory activity, whereas shRNA-mediated knockdown of Crx and Nrl resulted in reduced Kcnv2 promoter activity and low endogenous Kcnv2 mRNA expression in the retina. Site-directed mutagenesis of the CBS and NBS demonstrated that CBS2 is crucial for Kcnv2 promoter activity. We conclude that nucleotide changes in evolutionary conserved CBS could impact retina-specific expression levels of Kcnv2