Cluster analysis showing the expression changes of transcripts significantly affected by NELF depletion. Triplicate samples for each condition were averaged, and pairwise comparisons between conditions were performed. Shown are 241 transcripts that changed >1.5-fold in NELF-depleted cells, with a P-value <0.001 as compared with both untreated and mock-treated cells: untreated versus mock-treated (lane 1); untreated versus NELF-depleted (lane 2); mock-treated versus NELF-depleted (lane 3). Several genes of interest are labeled at right. Red indicates an increase in RNA levels, while green indicates a decrease. Values are given in log base 2 units, with the color bar shown at bottom right.

Pol II and NELF occupy the promoter-proximal regions of NELF target genes. ChIP material was immunoprecipitated with antibodies that recognize the Rpb3 subunit of Pol II or NELF-E. The percentage of input obtained in each ChIP sample is plotted for the genes designated, with positions given representing the center of each primer pair with respect to the transcription start site for that gene. ChIP signals are shown for NELF-unaffected genes eIF-5C and RpL3 (A); up-regulated genes Hsp70, mfas, oaf, and CG9008 (B); and down-regulated genes nocturnin, Mmp2, wun2, CG11739, egr, and TepII (C).

Depletion of NELF alters the chromatin environment at down-regulated genes. (A) Histone H3 ChIP signal at the promoters of NELF-unaffected genes RpL3 and eIF-5C; up-regulated genes Hsp70 and oaf; and down-regulated genes TepII and Tl. (B) Change in H3 ChIP signal between mock-treated and NELF-depleted cells as a fraction of mock-treated H3 ChIP signal. Median H3 ChIP signal change at down-regulated promoters (n = 16) is significantly different from at NELF-unaffected (n = 9) and up-regulated (n = 7) promoters (P < 0.0001). (C) H3-K4-me3 ChIP signals obtained with an antibody against H3-K4-me3 and expressed as a fraction of H3 ChIP signal are shown at promoters and downstream regions of RpL3, Hsp70, and TepII. (D) Change in H3-K4-me3 ChIP signal between mock-treated and NELF-depleted cells as a fraction of mock-treated H3-K4-me3 ChIP signal for 16 NELF-unaffected and up-regulated promoters and downstream regions, and 16 down-regulated promoters and downstream regions. Median H3-K4-me3 ChIP signal change at down-regulated promoters and downstream regions is significantly different from at NELF-unaffected and up-regulated promoters and downstream regions (P < 0.0001).

Regulation of gene expression by NELF, Pol II stalling, and chromatin. (A) Differing effects of NELF-RNAi upon expression driven by transiently transfected down-regulated or up-regulated promoters. S2 cells were transfected with vectors expressing firefly luciferase driven by the promoter regions of down-regulated genes Tl and TepII or up-regulated genes mfas and Hsp70. (B) Model for the dual roles of NELF and Pol II stalling in regulating gene expression. Pol II (rocket) initiates transcription, and NELF (oval) induces Pol II to stall in the promoter-proximal region. At up-regulated genes (left panel), NELF RNAi diminishes stalling such that polymerase moves into the gene and is replaced by newly recruited Pol II, thereby increasing transcriptional output. The promoter retains H3-K4 trimethylation and low nucleosome density. At down-regulated genes, NELF RNAi leads to a significant decrease in occupancy of the promoter-proximal region by stalled Pol II, which is associated with the establishment of a more repressive chromatin structure, including increased nucleosome occupancy of the promoter region, and a reduction in histone H3-K4 trimethylation (depicted as additional nucleosome and loss of asterisks). The increased nucleosome density further inhibits recruitment of additional Pol II and decreases transcription of these genes.

NELF target genes are involved in cellular responses to stimuli. A query of the Gene Ontology Database with a list of the 168 NELF target genes that have annotated Biological Processes reveals several classes that are significantly over-represented in this gene list (P-value cutoff <0.001). The number of genes in each Gene Ontology class, the percentage of total genes in that class that are NELF-regulated, and the associated P-values are given.

Depletion of NELF significantly alters Pol II distribution at down-regulated genes. (A) Pol II ChIP signal at the promoters of NELF-unaffected genes RpL3 and eIF-5C; up-regulated genes Hsp70 and oaf; and down-regulated genes TepII and Tl in cells that were either mock-treated or depleted of NELF using RNAi. (B) Change in Pol II ChIP signal between mock-treated and NELF-depleted cells as a fraction of mock-treated Pol II ChIP signal for nine NELF-unaffected promoters, seven up-regulated promoters, and 16 down-regulated promoters. Median Pol II ChIP signal change at down-regulated promoters (−70%) is significantly different from NELF-unaffected (−36%) and up-regulated (−38%) promoters (P < 0.0001; Kruskal-Wallis test). (C) Analysis of permanganate reactivity at NELF target genes in S2 cells. Lanes depict, from left to right: the A+G ladder used to determine the position of the promoter (shown by arrow), the permanganate reactivity of naked DNA as a control, and reactivity pattern of the indicated genes in S2 cells that were either mock-treated or depleted of NELF. (D) Analysis of permanganate reactivity in salivary glands from wild-type and NELF-D RNAi flies. Lanes from left to right depict the permanganate reactivity of naked DNA and of the indicated genes in salivary glands from either wild-type (control) or NELF-D-depleted flies.

Obstruction of promoter regions by nucleosomes in response to NELF depletion. (A) Histone H3 ChIP signal at the down-regulated gene TepII in mock-treated and NELF-depleted S2 cells, revealed using primer pairs spanning the TepII transcription start site. For comparison, major sites of permanganate reactivity indicating the locations of stalled Pol II are shown below. (B) Nucleosome locations at TepII are altered by depletion of NELF. Chromatin from untreated, NELF-depleted, and mock-treated cells was digested with MNase and PCR performed as above. (C) DNase I hypersensitivity at promoters of NELF target genes. Lanes depict, from left to right: the A + G ladder used for position determination, the reactivity of naked DNA as a control, and the DNase I hypersensitivity of the indicated genes in nuclei isolated from S2 cells that were either mock-treated or NELF-depleted. Major sites of permanganate reactivity indicating the locations of stalled Pol II at the rho, TepII, and Tl genes are shown at right.