Origin of RNA Polymerase II pause in eumetazoans: Insights from Hydra

Abstract

Multicellular organisms have evolved sophisticated mechanisms for responding to various developmental, environmental and physical stimuli by regulating transcription. The correlation of distribution of RNA Polymerase II (RNA Pol II) with transcription is well established in higher metazoans, however genome-wide information about its distribution in early metazoans, such as Hydra, is virtually absent. To gain insights into RNA Pol II-mediated transcription and chromatin organization in Hydra, we performed chromatin immunoprecipitation (ChIP)-coupled high-throughput sequencing (ChIP-seq) for RNA Pol II and Histone H3. Strikingly, we found that Hydra RNA Pol II is uniformly distributed across the entire gene body, as opposed to its counterparts in bilaterians such as human and mouse. Furthermore, correlation with transcriptome data revealed that the levels of RNA Pol II correlate with the magnitude of gene expression. Strikingly, the characteristic peak of RNA Pol II pause typically observed in bilaterians at the transcription start sites (TSSs) was not observed in Hydra. The RNA Pol II traversing ratio in Hydra was found to be intermediate to yeast and bilaterians. The search for factors involved in RNA Pol II pause revealed that RNA Pol II pausing machinery was most likely acquired first in Cnidaria. However, only a small subset of genes exhibited the promoter proximal RNP Pol II pause. Interestingly, the nucleosome occupancy is highest over the subset of paused genes as compared to total Hydra genes, which is another indication of paused RNA Pol II at these genes. Thus, this study provides evidence for the molecular basis of RNA Pol II pause early during the evolution of multicellular organisms.

Figure 1

Global RNA Pol II pausing is not evident in Hydra. Hydra RNA Pol II is uniformly distributed across the gene body. (A) Genome browser tracks of representative examples of loci showing uniform distribution of Hydra RNA Pol II in gene body. (B) Mean tag density of Hydra RNA Pol II calculated for whole gene and plotted for high, medium and least expressed genes (500 each). Levels of Hydra RNA Pol II correlate with magnitude of gene expression. (C) Normalized ChIP-sequencing enrichment for RNA Pol II from Drosophila, human, mouse, S. cerevisiae and Hydra. RNA Pol II pause was observed in mouse, human and Drosophila. RNA Pol II pause in Hydra was minimal (small peak at TSS as compared to other organisms) and the distribution of RNA Pol II in gene body was maximum.

Figure 2

Hydra RNA Pol II travelling ratio. Comparative RNA Pol II traveling ratio. RNA Pol II travelling ratio was calculated for genes from Drosophila, human, mouse, S. cerevisiae and Hydra. RNA Pol II travelling ratio was lowest in Hydra in all the examined species.

Figure 3

Evolution of NELF family members. Reported and putative NELF protein sequences were extracted from representative phyla and their phylogenetic analyses were performed using maximum likelihood method and their occurrence was summarized. (A) Phylogenetic tree of four classes of NELF members. All four classes of NELF members are shaded in distinct colours. NELF homologues identified in Hydra vulgaris Ind-Pune are highlighted in red colour font. Numbers on branch points denote bootstrap support values. (B) Summary table displaying the presence of NELF members in representative model organisms. Empty circle – absence; filled circle – presence and red filled circles represent homologues found in Hydra.

Figure 4

A select subset of genes exhibit RNA Pol II pausing in Hydra. (A) Average ChIP-seq profile of RNA Pol II occupancy over a subset of 235 promoters identified having paused RNA Pol II in Hydra. (B) RNA Pol II paused genes in Hydra are highly expressed as compared to the total transcripts levels. (C) Gene ontology analysis of RNA Pol II paused genes suggests enrichment of the pathways associated with gene expression regulation and translation.

Figure 5

+1 Nucleosome is tightly positioned on transcription start sites (TSSs) of RNA Pol II paused genes in Hydra. +1 nucleosome position was analysed on all the genes and RNA Pol II paused genes from Hydra. RNA Pol II paused genes exhibit highest +1 nucleosome occupancy.