Negative elongation factor (NELF) coordinates RNA polymerase II pausing, premature termination, and chromatin remodeling to regulate HIV transcription.

A barrier to eradicating HIV infection is targeting and eliminating latently infected cells. Events that contribute to HIV transcriptional latency include repressive chromatin structure, transcriptional interference, the inability of Tat to recruit positive transcription factor b, and poor processivity of RNA polymerase II (RNAP II). In this study, we investigated mechanisms by which negative elongation factor (NELF) establishes and maintains HIV latency. Negative elongation factor (NELF) induces RNAP II promoter proximal pausing and limits provirus expression in HIV-infected primary CD4(+) T cells. Decreasing NELF expression overcomes RNAP II pausing to enhance HIV transcription elongation in infected primary T cells, demonstrating the importance of pausing in repressing HIV transcription. We also show that RNAP II pausing is coupled to premature transcription termination and chromatin remodeling. NELF interacts with Pcf11, a transcription termination factor, and diminishing Pcf11 in primary CD4(+) T cells induces HIV transcription elongation. In addition, we identify NCoR1-GPS2-HDAC3 as a NELF-interacting corepressor complex that is associated with repressed HIV long terminal repeats. We propose a model in which NELF recruits Pcf11 and NCoR1-GPS2-HDAC3 to paused RNAP II, reinforcing repression of HIV transcription and establishing a critical checkpoint for HIV transcription and latency.

NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly.

The Negative Elongation Factor (NELF) is a transcription regulatory complex that induces stalling of RNA polymerase II (Pol II) during early transcription elongation and represses expression of several genes studied to date, including Drosophila Hsp70, mammalian proto-oncogene junB, and HIV RNA. To determine the full spectrum of NELF target genes in Drosophila, we performed a microarray analysis of S2 cells depleted of NELF and discovered that NELF RNAi affects many rapidly inducible genes involved in cellular responses to stimuli. Surprisingly, only one-third of NELF target genes were, like Hsp70, up-regulated by NELF-depletion, whereas the majority of target genes showed decreased expression levels upon NELF RNAi. Our data reveal that the presence of stalled Pol II at this latter group of genes enhances gene expression by maintaining a permissive chromatin architecture around the promoter-proximal region, and that loss of Pol II stalling at these promoters is accompanied by a significant increase in nucleosome occupancy and a decrease in histone H3 Lys 4 trimethylation. These findings identify a novel, positive role for stalled Pol II in regulating gene expression and suggest that there is a dynamic interplay between stalled Pol II and chromatin structure.

NELF and GAGA factor are linked to promoter-proximal pausing at many genes in Drosophila.

Recent analyses of RNA polymerase II (Pol II) revealed that Pol II is concentrated at the promoters of many active and inactive genes. NELF causes Pol II to pause in the promoter-proximal region of the hsp70 gene in Drosophila melanogaster. In this study, genome-wide location analysis (chromatin immunoprecipitation-microarray chip [ChIP-chip] analysis) revealed that NELF is concentrated at the 5' ends of 2,111 genes in Drosophila cells. Permanganate genomic footprinting was used to determine if paused Pol II colocalized with NELF. Forty-six of 56 genes with NELF were found to have paused Pol II. Pol II pauses 30 to 50 nucleotides downstream from transcription start sites. Analysis of DNA sequences in the vicinity of paused Pol II identified a conserved DNA sequence that probably associates with TFIID but detected no evidence of RNA secondary structures or other conserved sequences that might directly control elongation. ChIP-chip experiments indicate that GAGA factor associates with 39% of the genes that have NELF. Surprisingly, NELF associates with almost one-half of the most highly expressed genes, indicating that NELF is not necessarily a repressor of gene expression. NELF-associated pausing of Pol II might be an obligatory but sometimes transient checkpoint during the transcription cycle.

Transcription elongation controls cell fate specification in the Drosophila embryo.

The simple combinatorial rules for regulation of the sloppy-paired-1 (slp1) gene by the pair-rule transcription factors during early Drosophila embryogenesis offer a unique opportunity to investigate the molecular mechanisms of developmentally regulated transcription repression. We find that the initial repression of slp1 in response to Runt and Fushi-tarazu (Ftz) does not involve chromatin remodeling, or histone modification. Chromatin immunoprecipitation and in vivo footprinting experiments indicate RNA polymerase II (Pol II) initiates transcription in slp1-repressed cells and pauses downstream from the promoter in a complex that includes the negative elongation factor NELF. The finding that NELF also associates with the promoter regions of wingless (wg) and engrailed (en), two other pivotal targets of the pair-rule transcription factors, strongly suggests that developmentally regulated transcriptional elongation is central to the process of cell fate specification during this critical stage of embryonic development.

Molecular characterization of Drosophila NELF.

NELF and DSIF act together to inhibit transcription elongation in vitro, and are implicated in causing promoter proximal pausing on the hsp70 gene in Drosophila. Here, further characterization of Drosophila NELF is provided. Drosophila NELF has four subunits similar to subunits of human NELF. The amino acid sequences of NELF-B and NELF-D are highly conserved throughout their lengths, while NELF-A and NELF-E contain nonconserved regions inserted between conserved N- and C-terminal regions. Immunodepletion of NELF or DSIF from a nuclear extract desensitizes transcription in vitro to DRB. Immunodepletion of NELF also impairs promoter proximal pausing on the hsp70 promoter in vitro without affecting initiation. Chromatin immunoprecipitation analyses detect NELF at the promoters of the hsp70 and beta1-tubulin genes where promoter proximal pausing has been previously detected. Heat shock induction of hsp70 results in a marked decrease in NELF at the hsp70 promoter. Immunofluorescence analysis of polytene chromosomes shows extensive colocalization of the NELF-B and NELF-D subunits at hundreds of interbands. Neither subunit appears to be recruited to puffs. These results provide a foundation for genetic and biochemical analysis of NELF in Drosophila.