Jianlong Sun

Human Negative Elongation Factor Activates Transcription and Regulates Alternative Transcription Initiation

The human negative elongation factor (NELF) is a four-subunit protein complex that inhibits the movement of RNA polymerase II (RNAPII) at an early elongation stage in vitro. NELF-mediated stalling of RNAPII also attenuates transcription of a number of inducible genes in human cells. To obtain a genome-wide understanding of human NELF-mediated transcriptional regulation in vivo, we carried out an exon array study in T47D breast cancer cells with transient small interfering RNA knockdown of individual NELF subunits. Upon depletion of NELF-A, -C, or -E, the vast majority of NELF-regulated genes were down-regulated. Many of the down-regulated genes encode proteins that play key roles in cell cycle progression. Consequently, NELF knockdown resulted in significant reduction in DNA synthesis and cell proliferation. Chromatin immunoprecipitation showed that NELF knockdown led to dissociation of RNAPII from the promoter-proximal region of the cell cycle-regulating genes. This was accompanied by decreased histone modifications associated with active transcription initiation (H3K9Ac) and elongation (H3K36Me3), as well as reduced recruitment of the general transcription factor TFIIB and increased overall histone occupancy at a subset of the down-regulated promoters. Lastly, our study indicates that NELF regulates alternative transcription initiation of BSG (Basigin) gene by differentially influencing RNAPII density at the two neighboring exons at the 5′ end of the gene. Taken together, our data suggest a diverse transcriptional consequence of NELF-mediated RNAPII pausing in the human genome.

Mouse Cofactor of BRCA1 (Cobra1) Is Required for Early Embryogenesis

Background Negative elongation factor (NELF) is a four-subunit protein complex conserved from Drosophila to humans. In vitro biochemical and tissue culture-based studies have demonstrated an important role of NELF in controlling RNA polymerase II (Pol II) pausing in transcription. However, the physiological significance of NELF function is not clear due to the lack of any genetic systems for studying NELF. Principal Findings Here we show that disruption of the mouse B subunit of NELF (NELF-B), also known as cofactor of BRCA1 (Cobra1), causes inner cell mass (ICM) deficiency and embryonic lethality at the time of implantation. Consistent with the phenotype of the Cobra1 knockout (KO) embryos, knockdown of Cobra1 in mouse embryonic stem cells (ESCs) reduces the efficiency of colony formation and increases spontaneous differentiation. Cobra1-depleted ESCs maintain normal levels of Oct4, Nanog, and Sox2, master regulators of pluripotency in ESCs. However, knockdown of Cobra1 leads to precocious expression of developmental regulators including lymphoid enhancer-binding factor 1 (Lef1). Chromatin immunoprecipitation (ChIP) indicates that Cobra1 binds to the Lef1 promoter and modulates the abundance of promoter-bound RNA polymerase. Conclusions Cobra1 is essential for early embryogenesis. Our findings also indicate that Cobra1 helps maintain the undifferentiated state of mESCs by preventing unscheduled expression of developmental genes.

Deregulation of cofactor of BRCA1 expression in breast cancer cells

Cofactor of BRCA1 (COBRA1) is an integral component of the human negative elongation factor (NELF), a four‐subunit protein complex that inhibits transcription elongation. Previous in vivo work indicates that COBRA1 and the rest of the NELF complex repress estrogen‐dependent transcription and the growth of breast cancer cells. In light of the COBRA1 function in breast cancer‐related gene expression, we sought to examine regulation of COBRA1 expression in both established breast cancer cell lines and breast carcinoma tissues. We found that COBRA1 expression was inversely correlated with breast cancer progression, as tumor samples of patients who had distant metastasis and local recurrence expressed very low levels of COBRA1 mRNA when compared to those who were disease free for over 10 years (P = 0.0065 and 0.0081, respectively). Using both breast and prostate cancer cell lines, we also explored the possible mechanisms by which COBRA1 expression is regulated. Our results indicate that the protein abundance of COBRA1 and the other NELF subunits are mutually influenced in a tightly coordinated fashion. Small interfering RNA (siRNA) that targeted at one NELF subunit dampened the protein levels of all four subunits. Conversely, ectopic expression of COBRA1 in the knockdown cells partially rescues the co‐depletion of the NELF subunits. In addition, our study suggests that a post‐transcriptional, proteasome‐independent mechanism is involved in the interdependent regulation of the NELF abundance. Furthermore, a lack of COBRA1 expression in breast carcinoma may serve as a useful indicator for poor prognosis. J. Cell. Biochem. 103: 1798–1807, 2008.

Cofactor of BRCA1 modulates androgen-dependent transcription and alternative splicing.

Transcriptional activity of nuclear receptors (NRs) is influenced by a large number of coregulators that exert their actions predominantly at the transcription initiation step. Unlike most well-characterized NR coregulators, cofactor of BRCA1 (COBRA1), a subunit of the negative elongation factor (NELF), binds to estrogen receptor alpha (ERalpha) and modulates estrogen-dependent transcription by impeding the movement of RNA polymerase II (RNAPII) during the transcription elongation stage. Here we show that, in addition to ERalpha, COBRA1 also displays various degrees of affinity for several other NRs. In particular, COBRA1 binds strongly to androgen receptor (AR) via its ligand-binding domain (LBD). Small hairpin RNA (shRNA)-mediated reduction of endogenous COBRA1 enhances androgen-mediated transcription. The effect of COBRA1 knockdown can be rescued by a silent mutant COBRA1 that is refractory to the shRNA action. Using a reporter assay for alternative splicing, we also provide evidence for a role of COBRA1 in influencing the exon skipping/inclusion of nascent transcripts produced from an androgen-dependent promoter. These findings suggest that COBRA1 may coordinate multiple steps in ligand-dependent gene expression, which in turn ensures both the quantity and quality of hormone-stimulated gene products.

Genetic and Genomic Analyses of RNA Polymerase II-pausing Factor in Regulation of Mammalian Transcription and Cell Growth

Background: RNA polymerase II pausing is an important regulatory step in eukaryotic transcription. Results: Genetic ablation of Nelf-b leads to deregulation of pol II pausing and defects in cell growth and survival. Conclusion: Nelf-b plays important roles in multiple aspects of transcriptional regulation in mammalian genomes. Significance: Understanding the functional consequences of Nelf-mediated pol II pausing is important for linking transcription with biology. Many mammalian genes are occupied by paused RNA polymerase II (pol II) in the promoter-proximal region on both sides of the transcription start site. However, the impact of pol II pausing on gene expression and cell biology is not fully understood. In this study, we used a Cre-Lox system to conditionally knock out the b subunit of mouse negative elongation factor (Nelf-b), a key pol II-pausing factor, in mouse embryonic fibroblasts. We found that Nelf-b was associated with the promoter-proximal region of the majority of expressed genes, yet genetic ablation of Nelf-b only affected the steady-state mRNA levels of a small percentage of the Nelf-b-associated genes. Interestingly, Nelf-b deletion also increased levels of transcription start site upstream transcripts at multiple negative elongation factor-associated genes. The direct target genes of Nelf-b were highly enriched with those involved in the control of cell growth and cell death. Correspondingly, Nelf-b knock-out mouse embryonic fibroblasts exhibited slower progression from quiescence to proliferation, as well as in a cycling cell population. Furthermore, Nelf-b deletion also resulted in increased apoptosis. Thus, the genetic and genomic studies provide new physiological and molecular insight into Nelf-mediated pol II pausing.

Translational Initiation at a Non-AUG Start Codon for Human and Mouse Negative Elongation Factor-B.

Negative elongation factor (NELF), a four-subunit protein complex in metazoan, plays an important role in regulating promoter-proximal pausing of RNA polymerase II (RNAPII). Genetic studies demonstrate that the B subunit of mouse NELF (NELF-B) is critical for embryonic development and homeostasis in adult tissue. We report here that both human and mouse NELF-B proteins are translated from a non-AUG codon upstream of the annotated AUG. This non-AUG codon sequence is conserved in mammalian NELF-B but not NELF-B orthologs of lower metazoan. The full-length and a truncated NELF-B that starts at the first AUG codon both interact with the other three NELF subunits. Furthermore, these two forms of NELF-B have a similar impact on the transcriptomics and proliferation of mouse embryonic fibroblasts. These results strongly suggest that additional amino acid sequence upstream of the annotated AUG is dispensable for the essential NELF function in supporting cell growth in vitro. The majority of mouse adult tissues surveyed express the full-length NELF-B protein, and some contain a truncated NELF-B protein with the same apparent size as the AUG-initiated version. This result raises the distinct possibility that translational initiation of mouse NELF-B is regulated in a tissue-dependent manner.