1. Chromatin transcription and regulation in human cells
1.1 Key concepts of Pol II transcription regulation
As outlined above, Pol II undergoes an elaborate and repetitive cycle from initiation to recycling which is enabled and regulated by factors acting in trans with Pol II and the nascent RNA backbone 75,76. Multiple interconnected steps allow for control of when and to which extend transcriptional output is generated. These regulatory steps entail changes in chromatin accessibility 77,78, (co-) regulator recruitment 79, as well as allosteric changes 80 and post-translational modifications of factors involved in initiation, promoter-proximal pausing, elongation, termination and recycling 81. Post-translational modifications are reversible and allow for a dynamic code using regulation by writers, readers and erasers 82. Since the characterization of Pol II was initiated 50 years ago by Pierre Chambon and Robert Roeder
83-86, substantial advances have been made towards a mechanistic understanding of transcription and its regulation.
Figure 1. Key regulatory concepts of chromatin transcription in human cells.
(a) Genome organization. Left: 3D organization of chromatin in the cell nucleus. Middle: zoom to chromosome territories. Right: representation of the transcription cycle. Pol II is depicted in silver (the CTD of Pol II is not shown) and additional factors in dark grey. RNA and RNA-binding proteins (RBPs) are depicted in blue. Solid line represents DNA and nucleosomes (chromatin). Right schematic is adapted from Hantsche and Cramer 87. (b) Transcriptional states ordered by activity. The basal state varies between segments of the genome. The ground state of the majority of the genome is repressed (basal state ‘B’), while certain regions have a higher intrinsic activity (basal state ‘A’) such as promoters. Parts of the genome are strongly repressed (basal state ‘C’) such as pericentric heterochromatin. Right: negative factors repress transcription, while activators and positive co-factors increase transcriptional activity. Schematic is adapted from Burley and Roeder 88. (c) Pol II has an unstructured CTD. Disorder analysis (top) and schematic view (bottom) was kindly provided by Marc Böhning (MPI-bpc, Dept. of Molecular Biology) 89. (d) Simplified representation shows the layers of transcription regulation.
For details refer to main text. (e) Diagram illustrating the classes of genes (as boxes on the plus or minus DNA strand) encoding protein-coding and long noncoding RNAs: messenger (m) RNA in green; long intergenic noncoding (linc) RNA in purple; enhancer (e) RNA in red; antisense (as) RNA, upstream antisense (ua) RNA, convergent (con) RNA, and short intergenic noncoding (sinc) RNA in black. Sense TSS is marked by asterisk.
Promoter states (grey ovals) are associated with multiple gene classes, whereas enhancer states (red ovals) are only associated with genes encoding eRNAs. Top and bottom panels: boxes represent transcribed exons, solid lines represent introns.
For a detailed state-of-the-art picture of the individual steps of transcription the reader is referred to several excellent reviews with recent structure-function 87,90,91, biochemical, single-molecule imaging 92,93, or functional genomics insights 25,48,64,71,94,95. The following paragraphs highlight multiple layers and the dynamics of transcription regulation relevant to this work (Figure 1).
3D organization and chromatin accessibility. Transcriptional activity of a genomic region depends on its accessibility to pioneering factors, remodelers, and transcription factors 96. This is determined by chromatin compaction 41,97, DNA sequence 98, topology 99 and its modifications (of CpG islands) 100 either of which might restrict access to underlying DNA elements. It is further influenced by the composition and post-translational modifications of the histone octamers 78. Histones, particularly their accessible N-terminal tail region, can be methylated (me), acetylated (ac), phosphorylated, ubiquitinated, sumoylated, ADP ribosylated, propionylated, buryrylated, deaminated 78,101,102 and serotonylated 103 (histone code). The addition or removal of modifications, or deposition of specialized histone variants can reduce chromatin compaction, act as scaffolds to recruit transcription activators or repressors and thus, associate dynamically with certain transcription states (active, poised, repressed, silenced) 101 (Figure 1 b). On top of this, nucleosome positioning 41 and spacing is actively regulated by ATP-dependent chromatin-remodeling complexes which slide, exchange, and evict nucleosomes 40.
Repetitive carboxy-terminal domain (CTD) of POLR2A. Pol II is a 514 kDa enzyme consisting of 12 subunits 104,105.The largest subunit of Pol II, POLR2A (alias human Rpb1) has a large unstructured CTD which consists of a linker and 52 repeats with the consensus sequence YSPTSPS (Y: tyrosine, S: serine, P: proline, and T: threonine) 82,106,107 (Figure 1 c).
It serves as landing platform for transcription factors. The CTD of Pol II is hypophosphorylated when it is not bound to its template DNA 108,109. Beginning from initiation, cyclin-dependent kinases (CDKs) decorate the CTD linker 110,111 and repeats with phosphorylations 82 (Figure 1 d). These dynamic modifications recruit factors specific to each step of the transcription cycle 81,82 (CTD code). Upon recycling, the RNA is released, the CTD modifications are reset and Pol II is available for another round of transcription 112.
Concentration of factors. Binding of any factor to Pol II or nascent RNA backbone is dictated by both the concentration of the factor itself and the number of competing binding sites on the target 113. Each step of the transcription cycle requires a distinct set of factors which have to be provided and organized in space and time 17,114. An attractive model for local ‘caging’
and organization of multiple factors is liquid-liquid phase separation (LLPS) 115-117. Furthermore, LLPS might be a crucial for 3D nuclear organization 95. As it stands today, it is not entirely clear what the components (DNA 118, RNA 119, proteins) of transcription condensates are. However, recent reports show that proteins with low complexity intrinsically disordered regions like the CTD of POLR2A 89,120 or the histidine-rich domain in the cyclin subunit of P-TEFb 121 (chapter 2.1) have the potential to phase separate. Switches of the phosphorylation status of the Pol II CTD allow to drive or prevent LLPS dynamically 89,120. (Co-) regulators such as the Mediator complex. Regulators might act as activators or
activators 123 and the Mediator complex to direct Pol II to the correct genomic loci 124. Activators are characterized by a bipartite organization consisting of a sequence-specific DNA-binding domain and an activation domain 125. The activation domain contacts and recruits additional multi-protein complexes which are referred to as co-activators 126,127. A crucial co-activator is the Mediator complex which bridges dynamically between both co-activators and Pol II as part of the PIC 124.
Transition to productive elongation. After Pol II escapes the promoter, the RNA 5’ end is capped and Pol II enters a promoter-proximal window 68,69. Pol II traverses this window at a rate controlled by accessibility, concentration of factors, and regulators before its release into productive elongation 19,92 (chapter 2). Elongating Pol II is highly processive 128. Elongation occurs at different velocities along a gene 129,130 (chapter 3.1.1) and thus, coordinates co-transcriptional mechanisms such as RNA processing 71,131,132, or chromatin modifications by recruitment of modifiers 133. The process of co-transcriptional mechanisms might also affect the elongation velocity 63. In addition, dynamically formed RNA secondary structures might modulate elongation velocity due to co-transcriptional folding 134.
Termination and recycling. Efficient termination is important for maintaining the pool of free polymerases for re-initiation 112. Termination is coupled to RNA 3’ end processing and depends on the dissociation of elongation factors in concert with recruitment of termination factors 135. For recycling and re-initiation of Pol II and the transcription machinery, two nonexclusive models have been proposed: chromatin loop formation to bring the 3’ end to the vicinity of the TSS 136-138, or chromatin compartmentalization 139 for local caging of transcription factors 112.
If factors involved in the transcription cycle act independently, the informational output would be a simple summation of the effects of each individual factor listed above. However, fine-tuning of transcriptional output suggests an intensive crosstalk between molecular processes which remains to be characterized (Figure 1 d). Factors may synergize or antagonize the functions of each other, leading to a complex output dependent on the specific composition.