Role of negative elongation factor in regulation of gene expression and breast cancer progression

Negative elongation factor (NELF) is a four-subunit protein complex, which represses transcription elongation of RNA polymerase II (RNAPII) shortly after transcription initiation. NELF-mediated temporary stalling of RNAPII is alleviated by the counteracting positive elongation factor b (P-TEFb) that phosphorylates RNAPII and NELF itself before RNAPII is fully engaged in the productive elongation process. While these biochemical activities of NELF in the regulation of elongation have been extensively characterized, it remains obscure with regard to the impact of NELF on in vivo gene expression, and furthermore, the effect of NELF on human diseases is largely unknown.;In the current dissertation work, I have combined both gene-specific approaches and whole-genome scale analysis to study the in vivo function of NELF in regulation of gene expression in mammalian cells. I have found that the B subunit of NELF, or cofactor of BRCA1 (COBRA1), is a novel transcriptional corepressor of multiple steroid hormone receptors, including estrogen receptor alpha (ERalpha) and androgen receptor (AR). COBRA1 directly interacts with these receptors and attenuates their transcriptional activity in hormone-dependent gene expression (Chapter I).;Besides the gene-specific studies, I have also examined the effect of NELF, as a protein complex, in the regulation of gene expression in an unbiased manner by performing gene expression profiling experiment. Intriguingly, I found that efficient depletion of NELF from cells by small interference RNA (siRNA) led to down-regulation of a large number of genes that are involved in cell cycle progression. As revealed by chromatin-immunoprecipitation (ChIP) analysis, NELF is recruited to the promoter-proximal regions of these affected genes, and depletion of NELF reduces association of RNAPII at the same region, which is at least partially due to impaired transcription initiation. Therefore, it is evident from these results that NELF may act as either a positive or negative regulator of transcription under different gene contexts (Chapter II).;In addition to transcription, I have also investigated the effect of NELF on regulation of alternative splicing, which is one of the mRNA processing events that are tightly coupled with transcription elongation. Although NELF is unlikely a bona fide splicing factor, alternative splicing of several genes is indeed altered upon NELF depletion, which further demonstrates the multifunctional facets of NELF in the regulation of gene expression (Chapter I&II).;In light of the intimate connection between COBRA1 and ERalpha and the importance of estrogen signaling in breast cancer, I have participated in a collaborative investigation to understand the potential role of COBRA1 in breast cancer progression. As the first step, we examined COBRA1 expression in clinical samples from both tumor and normal tissues. We found that COBRA1 expression was inversely correlated with disease progression. Patients who had distant metastasis and local recurrence expressed very low levels of COBRA1 in the primary tumors. In line with the clinical data, we found that, when compared with the control knockdown cells, COBRA1 knockdown breast cancer cells had a much higher capability to grow as a xenograft in nude mice under extremely low level of estrogen. This suggests that COBRA1 reduction increases estrogen sensitivity in breast cancer cells, which offers a partial explanation to the clinical observation (Chapter III).;Taken together, my work has revealed a multifunctional characteristic of NELF in the regulation of gene expression, and it has also provided a functional link between regulation of transcription elongation and breast cancer progression.