Cheng-Fu Kao

Histone H2B Ubiquitylation Is Associated with Elongating RNA Polymerase II

ABSTRACT Rad6-mediated ubiquitylation of histone H2B at lysine 123 has been linked to transcriptional activation and the regulation of lysine methylation on histone H3. However, how Rad6 and H2B ubiquitylation contribute to the transcription and histone methylation processes is poorly understood. Here, we show that the Paf1 transcription elongation complex and the E3 ligase for Rad6, Bre1, mediate an association of Rad6 with the hyperphosphorylated (elongating) form of RNA polymerase II (Pol II). This association appears to be necessary for the transcriptional activities of Rad6, as deletion of various Paf1 complex members or Bre1 abolishes H2B ubiquitylation (ubH2B) and reduces the recruitment of Rad6 to the promoters and transcribed regions of active genes. Using the inducible GAL1 gene as a model, we find that the recruitment of Rad6 upon activation occurs rapidly and transiently across the gene and coincides precisely with the appearance of Pol II. Significantly, during GAL1 activation in an rtf1 deletion mutant, Rad6 accumulates at the promoter but is absent from the transcribed region. This fact suggests that Rad6 is recruited to promoters independently of the Paf1 complex but then requires this complex for entrance into the coding region of genes in a Pol II-associated manner. In support of a role for Rad6-dependent H2B ubiquitylation in transcription elongation, we find that ubH2B levels are dramatically reduced in strains bearing mutations of the Pol II C-terminal domain (CTD) and abolished by inactivation of Kin28, the serine 5 CTD kinase that promotes the transition from initiation to elongation. Furthermore, synthetic genetic array analysis reveals that the Rad6 complex interacts genetically with a number of known or suspected transcription elongation factors. Finally, we show that Saccharomyces cerevisiae mutants bearing defects in the pathway to H2B ubiquitylation display transcription elongation defects as assayed by 6-azauracil sensitivity. Collectively, our results indicate a role for Rad6 and H2B ubiquitylation during the elongation cycle of transcription and suggest a mechanism by which H3 methylation may be regulated.

H2B Ubiquitylation Plays a Role in Nucleosome Dynamics during Transcription Elongation

The monoubiquitylation of histone H2B has been associated with transcription initiation and elongation, but its role in these processes is poorly understood. We report that H2B ubiquitylation is required for efficient reassembly of nucleosomes during RNA polymerase II (Pol II)-mediated transcription elongation in yeast. This role is carried out in cooperation with the histone chaperone Spt16, and in the absence of H2B ubiquitylation and functional Spt16, chromatin structure is not properly restored in the wake of elongating Pol II. Moreover, H2B ubiquitylation and Spt16 play a role in each others regulation. H2B ubiquitylation is required for the stable accumulation of Spt16 at the GAL1 coding region, and Spt16 regulates the formation of ubiquitylated H2B both globally and at the GAL1 gene. These data provide a mechanism linking H2B ubiquitylation to Spt16 in the regulation of nucleosome dynamics during transcription elongation.

Epithelial cell adhesion molecule regulation is associated with the maintenance of the undifferentiated phenotype of human embryonic stem cells.

Human embryonic stem cells (hESCs) are unique pluripotent cells capable of self-renewal and differentiation into all three germ layers. To date, more cell surface markers capable of reliably identifying hESCs are needed. The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein expressed in several progenitor cell populations and cancers. It has been used to enrich cells with tumor-initiating activity in xenograft transplantation studies. Here, we comprehensively profile the expression of EpCAM by immunofluorescence microscopy, Western blotting, and flow cytometry using an anti-EpCAM monoclonal antibody (mAb) OC98-1. We found EpCAM to be highly and selectively expressed by undifferentiated rather than differentiated hESCs. The protein and transcript level of EpCAM rapidly diminished as soon as hESC had differentiated. This silencing was closely and exclusively associated with the radical transformation of histone modification at the EpCAM promoter. Moreover, we demonstrated that the dynamic pattern of lysine 27 trimethylation of histone 3 was conferred by the interplay of SUZ12 and JMJD3, both of which were involved in maintaining hESC pluripotency. In addition, we used chromatin immunoprecipitation analysis to elucidate the direct regulation by EpCAM of several reprogramming genes, including c-MYC, OCT-4, NANOG, SOX2, and KLF4, to help maintain the undifferentiation of hESCs. Collectively, our results suggest that EpCAM might be used as a surface marker for hESC. The expression of EpCAM may be regulated by epigenetic mechanisms, and it is strongly associated with the maintenance of the undifferentiated state of hESCs.

The SAGA coactivator complex acts on the whole transcribed genome and is required for RNA polymerase II transcription

The SAGA (Spt-Ada-Gcn5 acetyltransferase) coactivator complex contains distinct chromatin-modifying activities and is recruited by DNA-bound activators to regulate the expression of a subset of genes. Surprisingly, recent studies revealed little overlap between genome-wide SAGA-binding profiles and changes in gene expression upon depletion of subunits of the complex. As indicators of SAGA recruitment on chromatin, we monitored in yeast and human cells the genome-wide distribution of histone H3K9 acetylation and H2B ubiquitination, which are respectively deposited or removed by SAGA. Changes in these modifications after inactivation of the corresponding enzyme revealed that SAGA acetylates the promoters and deubiquitinates the transcribed region of all expressed genes. In agreement with this broad distribution, we show that SAGA plays a critical role for RNA polymerase II recruitment at all expressed genes. In addition, through quantification of newly synthesized RNA, we demonstrated that SAGA inactivation induced a strong decrease of mRNA synthesis at all tested genes. Analysis of the SAGA deubiquitination activity further revealed that SAGA acts on the whole transcribed genome in a very fast manner, indicating a highly dynamic association of the complex with chromatin. Thus, our study uncovers a new function for SAGA as a bone fide cofactor for all RNA polymerase II transcription.

Histone ubiquitylation and chromatin dynamics.

Histones are subject to several post-translational modifications, which act to regulate gene expression and other processes on the DNA template. One such modification is the addition of a single ubiquitin moiety, which has been reported to influence chromatin dynamics and exhibit cross-talk with other histone modifications. Mono-ubiquitylation of H2B has been reported in eukaryotes as divergent as budding yeast, flies and humans, and is linked to transcriptional activation and gene silencing. Furthermore, ubiquitylation of H2A is also important for transcriptional repression in higher eukaryotes, and both histones play key roles in DNA repair. In this review, we will give an overview of the enzymes important for ubiquitylation and deubiquitylation of the various histone species, before examining the role of ubiquitylated histones in shaping the chromatin landscape and thus controlling the accessibility of DNA to effector proteins, through putative roles in promoting histone-histone interactions and stabilizing the structure of nucleosomes. We will finally discuss other processes reported to involve ubiquitylation of histones, including DNA repair, recombination and mRNA processing, underlining the diverse actions of these modifications.

DNA methylation and histone modification regulate silencing of OPG during tumor progression.

The identification of molecules that are down‐regulated in malignant phenotype is important for understanding tumor biology and their role in tumor suppression. We compared the expression profile of four normal nasal mucosal (NNM) epithelia and a series of nasopharyngeal cancinoma (NPC) cell lines using cDNA microarray and confirmed the actual expression of the selected genes, and found osteoprotegerin (OPG) to be ubiquitously deficient in NPC cells. We also found OPG to be down‐regulated in various cancer cell lines, including oral, cervical, ovarian, lung, breast, pancreas, colon, renal, prostate cancer, and hepatoma. Administration of recombinant OPG (rOPG) brought about a reduction in cancer cell growth through apoptotic mechanism. We generated eleven monoclonal antibodies (MAbs) against OPG to study OPGs expression and biological functions in cancer cells. OPG was detected in the tumor stromal regions, but not in the cancer cell per se in surgical specimens of liver cancer. Quantitative reverse transcription‐polymerase chain reaction (Q‐RT‐PCR) revealed that OPG was down‐regulated in NPC tissues compared with normal nasal polyp (NNP) tissues. In addition, we showed OPG silencing to be associated with promoter methylation as well as histone modifications. In OPG‐silenced cancer cell lines, the OPG gene promoter CpG dinucleotides were highly methylated. Compared to normal cells, silenced OPG gene in cancer cells were found to have reduced histone 3 lysine 4 tri‐methylation (H3K4me3) and increased histone 3 lysine 27 tri‐methylation (H3K27me3). Taken together, these results suggest that OPG silencing in carcinoma cancer cells occurs through epigenetic repression. J. Cell. Biochem. 108: 315–325, 2009.

Interplay between SIN3A and STAT3 mediates chromatin conformational changes and GFAP expression during cellular differentiation.

Background Neurons and astrocytes are generated from common neural precursors, yet neurogenesis precedes astrocyte formation during embryogenesis. The mechanisms of neural development underlying suppression and de-suppression of differentiation- related genes for cell fate specifications are not well understood. Methodology/Principal Findings By using an in vitro system in which NTera-2 cells were induced to differentiate into an astrocyte-like lineage, we revealed a novel role for Sin3A in maintaining the suppression of GFAP in NTera-2 cells. Sin3A coupled with MeCP2 bound to the GFAP promoter and their occupancies were correlated with repression of GFAP transcription. The repression by Sin3A and MeCP2 may be an essential mechanism underlying the inhibition of cell differentiation. Upon commitment toward an astrocyte-like lineage, Sin3A- MeCP2 departed from the promoter and activated STAT3 simultaneously bound to the promoter and exon 1 of GFAP; meanwhile, olig2 was exported from nuclei to the cytoplasm. This suggested that a three-dimensional or higher-order structure was provoked by STAT3 binding between the promoter and proximal coding regions. STAT3 then recruited CBP/p300 to exon 1 and targeted the promoter for histone H3K9 and H3K14 acetylation. The CBP/p300-mediated histone modification further facilitates chromatin remodeling, thereby enhancing H3K4 trimethylation and recruitment of RNA polymerase II to activate GFAP gene transcription. Conclusions/Significance These results provide evidence that exchange of repressor and activator complexes and epigenetic modifications are critical strategies for cellular differentiation and lineage-specific gene expression.

In vivo assays to study histone ubiquitylation

The importance of histone acetylation, phosphorylation, and methylation in transcription and other DNA-mediated processes is now well established. Histones are also ubiquitylated, but in contrast to the majority of ubiquitylated proteins, ubiquitylated histones are not generally targeted for degradation and may play roles similar to those of other histone modifications. Antibodies against acetylated histones have provided unique insights into the regulation, distribution, and cellular roles of these modified histones. In this report, we describe methods to identify ubiquitylated histones in budding yeast and HeLa cells. We provide protocols to detect ubiquitylated histones that are based on a combination of in vivo genetic and immunological assays. These methods should provide relatively simple and useful tools to study the global regulation of this important but poorly understood histone modification.

LHX2 regulates the neural differentiation of human embryonic stem cells via transcriptional modulation of PAX6 and CER1

The LIM homeobox 2 transcription factor Lhx2 is known to control crucial aspects of neural development in various species. However, its function in human neural development is still elusive. Here, we demonstrate that LHX2 plays a critical role in human neural differentiation, using human embryonic stem cells (hESCs) as a model. In hESC-derived neural progenitors (hESC-NPs), LHX2 was found to be expressed before PAX6, and co-expressed with early neural markers. Conditional ectopic expression of LHX2 promoted neural differentiation, whereas disruption of LHX2 expression in hESCs significantly impaired neural differentiation. Furthermore, we have demonstrated that LHX2 regulates neural differentiation at two levels: first, it promotes expression of PAX6 by binding to its active enhancers, and second, it attenuates BMP and WNT signaling by promoting expression of the BMP and WNT antagonist Cerberus 1 gene (CER1), to inhibit non-neural differentiation. These findings indicate that LHX2 regulates the transcription of downstream intrinsic and extrinsic molecules that are essential for early neural differentiation in human.

Flickin' the ubiquitin switch The role of H2B ubiquitylation in development

The reversible ubiquitylation of histone H2B has long been implicated in transcriptional activation and gene silencing. However, many questions regarding its regulation and effects on chromatin structure remain unanswered. In addition, while several studies have uncovered an involvement of this modification in the control of certain developmental processes, a more general understanding of its requirement is lacking. Herein, we present a broad overview of the pathways known to be regulated by H2B ubiquitylation, while drawing parallels between findings in disparate organisms, in order to facilitate continued delineation of its spatiotemporal role in development. Finally, we integrate the findings of recent studies into how H2B ubiquitylation affects chromatin, and cast an eye over emerging areas for future research.