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Paf1C regulates RNA polymerase II progression by modulating elongation rate

Significance The factors that regulate RNA polymerase II (Pol II) elongation rate and processivity are poorly understood. Here, we show that the Paf1 complex (Paf1C) modulates Pol II elongation rate, and loss of Paf1 results in accumulation of Ser5P in the first 20 to 30 kb of gene bodies, coinciding with reduction of histone H2B ubiquitylation specifically within this region. Moreover, reduced elongation rates provoked by Paf1 depletion resulted in defects in Pol II processivity and premature termination of transcription. Paf1 ablation did not impact the recruitment of other key elongation factors, suggesting that Paf1C function may be mechanistically distinguishable from each of these factors. Our data pinpoint Paf1C as a key modulator of Pol II elongation rates across mammalian genes.

C2C12 were infected with virus plus polybrene (1:2000; 8 mg/ml polybrene stock) for 2 days. Cells were selected with 2 μg/ml puromycin for 4 days. Expression of 2xFlag-2xHA-sgRNA-resistant-Paf1 was validated using anti-HA antibodies by immunofluorescence (Chromotek, Cat#7C9). Next, lentivirus expressing Cas9 and an sgRNA directed against exon 1 of Paf1 was generated as described above, and the endogenous Paf1 gene was targeted by infection of this lentivirus with 4 μg/ml polybrene in the presence of 1 μg/ml Dox. Cells were selected with 10 μg/ml Blasticidin for 4 days, and single cell-derived clones were isolated. Clones were screened by western blotting after removal Dox for 2-4 days. Clones lacking expression of endogenous Paf1 were further validated by PCR and DNA sequencing to ensure that deletion of both alleles resulted in frame shift and protein termination while cells simultaneously expressed the tagged Paf1 transgene (SI Appendix, Fig. S1A).

Antibodies
All antibodies used in this study are listed in Table S2 (SI Appendix).

RNAi
All siRNAs were synthesized by Dharmacon or Qiagen. Six hours before transfection, 1x10 5 C2C12 cells were seeded in 6-well plates and siRNAs were transfected into cells using Lipofectamine RNAiMAX (Invitrogen), at a final concentration of 50 nM. Cells were harvested 48 h post transfection. siRNA sequences are listed in SI Appendix, Table   S1.

Western blotting
Western blotting was performed as described previously (1) with minor modifications.

Chromatin immunoprecipitation (ChIP), ChIP-seq, and MNase-seq
ChIP was performed as described (3), followed by real-time quantitative PCR as described (4). All primer sequences are listed in SI Appendix, Table S1. ChIP-seq and MNase-seq experiments were performed as described (4).

DRB treatment
For Proseq experiments, DRB (Sigma, Cat# D1916) in DMSO was added to the media at a final concentration of 100 M for indicated times (5 min, 15 min, and 30 min), and the cells were quickly washed twice with cold PBS and collected.

Isolation of nuclei and Proseq
Nuclei were isolated from C2C12 cell as described (5)

Preparation of soluble chromatin
Chromatin-bound proteins were prepared through micrococcal nuclease digestion as described (7)

Mass spectrometry (MS)
Mass spectrometric identification of proteins associated with Paf1 in solubilized chromatin was carried out essentially as described (8).

Bioinformatics
All analyses in this study were performed using the UCSC mm9 mouse genome as reference.

RNA-seq analysis
For RNA-seq analysis, paired-end reads were aligned using STAR aligner. Reads that were not properly paired, non-uniquely aligned, or potential PCR duplicates were removed, and the remaining reads were used for subsequent analysis. DESeq2 (1.10.1) was used to identify differentially expressed mRNA. Only genes that had both a DESeq2 adjusted P value < 0.05 and a log 2 (FPKM) of at least 1 (fold change = 2) in either direction were considered as differentially expressed genes. Scatter plot in SI Appendix, Fig. S7A was plotted using plotMA function in R and replicate correlation analyses in SI Appendix, Fig. S7B was plotted using pheatmap package in R.

ATAC-seq analysis
For ATAC-seq analysis, raw reads were aligned to the mm9 reference genome using Bowtie2. PCR duplicates were removed with Picard. Mitochondrial reads were removed prior to downstream analysis, and the remaining reads were corrected to account for the 9 bp insert introduced by the Tn5 transposase by offsetting the 5' ends by either +4 (for plus strand) or -5 (for minus strand) as described previously (1).

ChIP-seq, MNase-seq, and Proseq analysis
Reads were aligned using Bowtie2. Only reads that were properly paired and uniquely aligned were used for subsequent processing. Potential PCR duplicates were removed using Picard Suite. Reads for ChIP-seq and Proseq data were normalized per kilobase per million for data visualization in IGV.

Metagene and Scatter plots
Metagene plots in Fig. 3C, 4F, 5D, and SI Appendix, S7E and S8E were calculated and plotted using deepTools (3.1.2) (9) in Galaxy platform (https://usegalaxy.org/). Briefly, bigwigCompare tool was used to compare two normalized bigwig files to obtain the log 2 ratios. Reference-point mode was used to compute Matrix using computeMatrix tool, the values for each normalized bigwig file (or log 2 ratio of comparison of two bigwig files) were calculated around genomic regions as described in Figure legends, and any values after this region endpoint were discarded. Output from the computeMatrix tool was subsequently used to generate profile plots using the plotProfile tool of deepTools.
The ngs.plot suite (https://github.com/shenlab-sinai/ngsplot) was used to make all the other metagene plots besides above mentioned plots. For Proseq, read1 of each pairedend read was extracted to make strand-specific metagene plots using ngs.plot suite, and the extreme 5% values were removed.
Scatter plots of log 2 fold-changes of FPKM of two datasets in Fig. 4G, SI Appendix, S8B and S8C were calculated and generated in two regions (TSS to 20 kb downstream of TSS, and 20 kb downstream of TSS to TES) using R (v3.4.3).

Heatmaps
Both paired-end or single reads were aligned using Bowtie2 with built-in mouse mm9 genome index in Galaxy platform, and bamCoverage tool (within deepTools suite) was used to generate a coverage bigwig file from a given BAM file using 50 bp bin size and normalized to reads per kilobase per million. PCR duplicates were removed. TSScentered heatmaps were calculated and plotted using computeMatrix and plotHeatmap tools for the region spanning -2 kb upstream of TSS to +2 kb downstream of TSS, ranked by descending Paf1 ChIP-seq occupancy signal or -5 kb upstream of TSS to +100 kb downstream of TSS, ranked by gene length of non-overlapping expressed genes (n=5846).
Ratio heatmaps were calculated using deepTools BigwigCompare. Corresponding heatmaps were calculated based on descending Paf1 occupancy or gene length as above mentioned regions. For RNA-seq (in SI Appendix, Fig. S7 C and D) and Proseq data (in Fig. 5 A and C), ratio heatmaps were plotted separately for both forward (n=3023) and reverse strands (n=2745). Correlation heatmaps for replicates were generated using deepTools multiBamSummary, plotting coverage for consecutive non-overlapping bins of 1 kb of size in SI Appendix, Fig. S2B, S2E and S8A.

Calculation of transcription rates using a hidden Markov model (HMM)
After quality examination and adaptor trimming with FastQC and Trimmomatic, Proseq reads were aligned to mm9 reference genome using STAR with a default strandspecific mode. To calculate elongation rates in myoblasts before and after Paf1 depletion, we selected 1708 expressed genes (FPKM>1) with gene lengths >70 kb, given that mammalian elongation rates have been estimated at ~2 kb/min, and Pol II should not be and 15min-30min were deduced based on the distance travelled over these two time intervals.

Software and Algorithms
All software and algorithms used in this study are listed in SI Appendix, Table S3.

Accession numbers
All large-scale data sets have been deposited in Gene Expression Omnibus (GEO), and the accession number for these datasets in GEO is GSE116169. Paf1 ChIP-seq, H3K36me3 ChIP-seq and 3READs-seq data were published previously GSE72574.