RNA polymerases as moving barriers to condensin loop extrusion

“, trapped at transcription start sites by rifampicin) are directional barriers to condensin translocation, akin to CTCFs as directional barriers to cohesin in eukaryotes (4), or an RNAPindependent mechanism generates the “baseline asymmetry” of chromosome juxtaposition at loci outside of rRNA operons.”

“One can hypothesize that RNAP has a directional effect on condensin translocation, like the effect of CTCF proteins on cohesin in eukaryotes (4).”

“…do not have coupled transcription and translation, there is transcription-coupled splicing, and the spliceosome is of similar size to the ribosome (58), making the whole complex of a globular diameter > 70 nm. Future experiments will be needed to test whether SMCs can bypass steric barriers…”

“Rudner [2], Leonid A.”

“tclockwise(i) = tclockwise,operon(i) + tclockwise,rRNA(i) + tclockwise,tRNA(i) (3)”

“4A, right), when a translocating condensin encounters a transcribing RNAP, condensin is stalled and pushed back to the transcription termination site (condensin has a very low stall force measured in vitro (10) compared to RNAP (50)).”

“Although the size of isolated eukaryotic RNAPs are comparable to their bacterial counterparts [ ∼ 10 – 15 nm in diameter (56)], sizes of whole elongating transcription complexes in eukaryotes can be considerably larger.”

“Nevertheless, since SMC and CTCF interactions in eukaryotes play a regulatory role in gene expression (61), it is of interest to explore the potential role of SMC–transcription interactions in gene regulation in both bacteria and eukaryotes.”

“S8B), and there are relatively few highly transcribed genes (47); the average RNAP density is 0.”

“4 A, Right), when a translocating condensin encounters a transcribing RNAP, condensin is stalled and pushed back to the transcription termination site [condensin has a very low stall force measured in vitro (10) compared to RNAP (53)].”

“If eukaryotic RNAPs turn out as permeable to SMC as bacterial RNAPs, transcription in eukaryotes may not have a major effect on chromosome organization by loop extrusion, as compared to other molecules that specifically (and directionally) impede loop extrusion, like CTCF (20, 60).”

“4 A, Right), when a translocating condensin encounters a transcribing RNAP, condensin is stalled and pushed back to the transcription termination site [condensin has a very low stall force measured in vitro (10) compared to RNAP (53)].”

“Mirny [1,4,5]”

“This idea emerged from the observation that cohesin SMCs are enriched at sites of convergent transcription in yeast (50, 51) and in mammalian cells (35); this is further supported by other experiments demonstrating localization of cohesin mediated by transcription in cells (32) and in vitro (52).”

“Permeability of RNAPs to loop extrusion also suggests that SMCs should be able to effectively bypass other large steric barriers, such as nucleosomes in eukaryotes [consistent with experimental evidence, where nucleosome depletion does not affect condensin’s ability to form a mitotic chromosome (68) and a recent prepr…”

“Previous studies have suggested that a passive SMC ring can be pushed by transcribing RNAP (34, 50).”

“This idea emerged from the observation that cohesin SMCs are enriched at sites of convergent transcription in yeast (50, 51) and in mammalian cells (35); this is further supported by other experiments demonstrating localization of cohesin mediated by transcription in cells (32) and in vitro (52).”

“This idea emerged from the observation that cohesin SMCs are enriched at sites of convergent transcription in yeast (50, 51) and in mammalian cells (35); this is further supported by other experiments demonstrating localization of cohesin mediated by transcription in cells (32) and in vitro (52).”

“SMC complexes are characterized by a 3-part ring, composed of a dimer of SMC subunits, each with an ATPase domain and a long coiled-coil domain; a kleisin linker which closes the ring; and accessory proteins which bind to the linker to perform specific functions, depending on the organism (1).”

“Evidence also suggests that loop extrusion by cohesin SMCs underlies the formation of chromosomal domains during interphase (16 – 20).”

“Permeability of RNAPs to loop extrusion also suggests that SMCs should be able to effectively bypass other large steric barriers, such as nucleosomes in eukaryotes [consistent with experimental evidence, where nucleosome depletion does not affect condensin’s ability to form a mitotic chromosome (68) and a recent prepr…”

“While the molecular details of this process are yet to be fully understood (14, 15), loop extrusion appears to be a mechanism that can explain a wealth of chromosomal phenomena in eukaryotes and bacteria.”

“2 ATP/cohesin/s (66, 67), whereas yeast condensins have higher rates of ∼1.”

“This idea emerged from the observation that cohesin SMCs are enriched at sites of convergent transcription in yeast (50, 51) and in mammalian cells (35); this is further supported by other experiments demonstrating localization of cohesin mediated by transcription in cells (32) and in vitro (52).”

“While the molecular details of this process are yet to be fully understood (14, 15), loop extrusion appears to be a mechanism that can explain a wealth of chromosomal phenomena in eukaryotes and bacteria.”

“subtilis, as reported (7, 21), contacts from DNA segments close in the linear genome sequence, as in all organisms, give rise to the primary interaction diagonal, which extends from the bottom left to top right of the map (see Fig.”

“Predictions of the permeable moving-barriers mechanism can also be tested by single-molecule experiments similar to those that observed ATP-dependent condensin translocation (9) and loop extrusion (10) in vitro.”

“2 ATP/cohesin/s (66, 67), whereas yeast condensins have higher rates of ∼1.”

“This idea emerged from the observation that cohesin SMCs are enriched at sites of convergent transcription in yeast (50, 51) and in mammalian cells (35); this is further supported by other experiments demonstrating localization of cohesin mediated by transcription in cells (32) and in vitro (52).”

“This finding argues that dimerization (or oligomerization) of condensin may be required to obtain 2 distinct motor activities, consistent with previous experimental evidence and theoretical models (8, 14, 39, 40), or that a single 1-sided condensin dynamically switches directions of translocation, leading to apparent…”

“Plausible mechanisms of SMC and RNAP interaction must solve the following puzzle: How can condensin’s effective speed of translocation [measured at >800 bp/s via Hi-C and ChIP-seq (8)] be so strongly attenuated (>20-fold) by RNAP transcription [which moves at 40–90 bp/s (49)], depending only on the relative orientatio…”

“Amazingly, DNA juxtaposition extends from origin to terminus, generating a single 4-Mb “loop” (7, 21, 22).”

“1 RNAP/kb for most genes (48), in contrast with ∼10 RNAP/kb for rRNA genes (48) in our growth conditions (SI Appendix, section 4.”

“The maximum rate of condensin extrusion (vmax) based on the genome annotation model is related to the average rate of extrusion (vavg) by: vavg = vmax 1 Narm ∑Narm i=1 ηi , (12)”

“subtilis are co-oriented with replication (37), we posited that transcription could account for the tilt of the secondary diagonal by slowing down condensin translocation (Fig.”

“trapped at transcription start sites by rifampicin) are directional barriers to condensin translocation, akin to CTCFs as directional barriers to cohesin in eukaryotes (4), or an RNAP independent mechanism generates the “baseline asymmetry” of chromosome juxtaposition at loci outside of rRNA operons.”

“Permeability of RNAPs to loop extrusion also suggests that SMCs should be able to effectively bypass other large steric barriers, such as nucleosomes in eukaryotes [consistent with experimental evidence, where nucleosome depletion does not affect condensin’s ability to form a mitotic chromosome (68) and a recent prepr…”

“A secondary diagonal, which runs perpendicular to the primary, typical of many bacterial Hi-C interaction maps (7, 22, 27), indicates a symmetric juxtaposition of 2 chromosome arms about the parS sites located next to the origin of replication (ori) (Fig.”

“…maintaining DNA in a topological embrace [with mechanisms like “ inchworm, ” “ pumping, ” “ seg-ment-capture, ” and “ shackled-walker ” (9, 14, 25, 62 – 64)]; or 2) the SMC rings transiently open (65), disengage the topological embrace of the DNA, and reengage after passing the steric barrier…”

“Of particular interest, a recent preprint has shown that purified yeast condensins that extrude loops on DNA can bypass one another with short pauses of ∼7 s (59); this is similar to the time we measured for bacterial condensins to bypass elongating transcripts.”

“…using chromosome conformation capture (Hi-C) and chromatin immunoprecipitation combined with deep sequencing (ChIP-seq) have shown that condensins are preferentially loaded onto chromosomes via ParB proteins bound at parS sites located primarily adjacent to the origin of replication (23 – 25).”

“In vivo experiments recently showed that translocation by condensin is an active process (8, 28): Condensin complexes travel processively and bidirectionally away from the parS loading site, in a manner that appears to be ATP-dependent, at speeds exceeding 800 bp/s (29).”

“Permeability of RNAPs to loop extrusion also suggests that SMCs should be able to effectively bypass other large steric barriers, such as nucleosomes in eukaryotes [consistent with experimental evidence, where nucleosome depletion does not affect condensin’s ability to form a mitotic chromosome (68) and a recent prepr…”

“5to 3-kb bacterial operon generates RNA of physical dimensions up to ∼60 × 30 × 10 nm (57).”

“As an example, recent studies provide strong evidence that transcription can affect genome structure and SMC action (31 – 36).”

“We generated a strain in which the sole copy of the β′ (beta prime) subunit of RNAP was fused to yellow fluorescent protein (YFP) and an SsrA tag (β′-YFP-SsrA) and could be conditionally targeted for degradation (46) (SI Appendix, Fig.”

“Permeability of RNAPs to loop extrusion also suggests that SMCs should be able to effectively bypass other large steric barriers, such as nucleosomes in eukaryotes [consistent with experimental evidence, where nucleosome depletion does not affect condensin’s ability to form a mitotic chromosome (68) and a recent prepr…”

“Tcounter−clockwise(j− 1) = Tcouter−clockwise(j) + fmix 2 · tclockwise(i) + (1− fmix) 2 · tcounter−clockwise(j), (11)”

“embrace [with mechanisms like “inchworm,” “pumping,” “segment-capture,” and “shackled-walker” (9, 14, 25, 62–64)]; or 2) the SMC rings transiently open (65), disengage the topological embrace of the DNA, and reengage after passing the steric barrier [closer to the “walker”/“rock-climber” models (39, 40)].”

“In a strain with a parS site close to a large cluster of rRNA operons (parS+26o), adding rifampicin to exponentially growing cells, which inhibits the transition from transcription initiation to elongation (42), results in a partial straightening out of the secondary diagonal as shown previously (8, 28) (Fig.”

“Once loaded, condensins progress away from the parS sites (8, 26) along both chromosomal arms, thus juxtaposing them (7, 21), resulting in the characteristic “X”-shaped pattern on the Hi-C maps of many bacteria (27).”

“Single-molecule studies provide support for this or a similar mechanism by demonstrating that budding yeast condensin SMCs are mechano-chemical motors that can translocate along DNA (9), extrude DNA loops (10), and actively compact DNA (11 – 13).”

“Rifampicin is a well-characterized transcription inhibitor that prevents the transition from transcription initiation to elongation, but does not prevent binding of RNAP to DNA (42); moreover, RNAP molecules that have started to synthesize RNA will continue to synthesize their RNA until they reach the transcription te…”

“In vivo experiments recently showed that translocation by condensin is an active process (8, 28): Condensin complexes travel processively and bidirectionally away from the parS loading site, in a manner that appears to be ATP-dependent, at speeds exceeding 800 bp/s (29).”

“ηi =  γ if condensin crosses a gene convergent with transcription ρ if condensin crosses an rRNA locus convergent with transcription 1 otherwise (13)”

“Mirny [1,4,5]”

“Single-molecule studies provide support for this or a similar mechanism by demonstrating that budding yeast condensin SMCs are mechano-chemical motors that can translocate along DNA (9), extrude DNA loops (10), and actively compact DNA (11 – 13).”

“Accordingly, it remains a possibility that other DNA motifs or processes, correlated with gene orientations, influence condensin speeds (38).”

“This finding argues that dimerization (or oligomerization) of condensin may be required to obtain 2 distinct motor activities, consistent with previous experimental evidence and theoretical models (8, 14, 39, 40), or that a single 1-sided condensin dynamically switches directions of translocation, leading to apparent…”

“…DNA in a topological embrace [with mechanisms like “ inchworm, ” “ pumping, ” “ seg-ment-capture, ” and “ shackled-walker ” (9, 14, 25, 62 – 64)]; or 2) the SMC rings transiently open (65), disengage the topological embrace of the DNA, and reengage after passing the steric barrier [closer…”

“We note, however, that this does not preclude the possibility that a single SMC complex performs the 2 motor activities: For instance, an anchored “1-sided” SMC extruder can alternate the anchoring site and the “DNA reeling arm,” effectively performing 2-sided extrusion with uncoupled kinetics (41); this will be a top…”

“Alongside these two major mechanisms, additional processes (pairing of homologs and sisters[80,81]), transcription[52,59,86] and affinities between specific genomic regions mediated by specific molecular complexes (polycomb[91], splicing factors[89], etc.”

“The symmetry of the juxtaposed chromosome arms implies that bSMC should be a two-sided LEF (Brandão et al., 2019; Wang et al., 2017) .”

“This is reminiscent of the condensin-polymerase antagonism reported for bacteria ( 34 ) and flies ( 33 ) and inferred by super-resolution imaging in mESCs ( 59 ).”

“Because previous studies showed that two motor activities of the same LEF are independent of each other, we allowed continued extrusion by a motor subunit, even if the other subunit’s translocation was blocked 9,10,29 .”

“However, a study in bacteria suggests that a related chromatin “ring” (condensin II complex) recovers quickly from the encounters with the elongating RNAP machinery [389].”

“Previous studies have shown that condensin and RNA polII can interplay (Brandão et al., 2019) and it has been shown in yeast that cohesin could be pushed by the transcription machinery (Lengronne et al., 2004).”

“This may explain the accumulation of condensins near transcription 8,22,23 and replication sites 24 .”

“This suggests that the causal arrow between looping and transcription might point both ways: DNA loop formation may contribute to increased transcription of target genes, but very high levels of transcription could contribute to the weakening loops by antagonizing loop extrusion, as observed previously (Busslinger et…”

“subtilis consistent with bsSMCRNAP collisions forcing bsSMC backwards along DNA (61).”

“The ability of loop-extruding SMCs to bypass each other ( 9 ) or large DNA-bound obstacles ( 68 ) has been observed in single-molecule experiments, and bypassing of bacterial SMCs was revealed using engineered bacterial chromosomes ( 69 ).”

“Recent biophysical studies revealed that nucleosomes and other protein complexes restrict cohesin translocation ( 18 ), suggesting that transcriptional processes and chromatin remodelers could promote the cohesin translocation along the DNA strand ( 18 , 19 ).”

“Meanwhile, our observation that local punctate interactions change around sites where gene expression changes echo previous evidence that collisions between loop extrusion and transcription can influence local contact patterns (Brandao et al , 2019).”

“However, it has been shown that HEG (that results in strong TIDs) represent permissive barriers to cohesin loop extrusions in other species such as Bacillus subtilis 43 .”

“subtilis suggest that loop extrusion by SMC complexes is mediated by two “handcuffed” rings (Brandão et al., 2019; Wang et al., 2015).”

“Notably, bacterial SMC complex speed 44 in vivo reduces through a head on conflict with transcription [68, 72, 74].”

“We assume uniform LEF extrusion speed throughout the genome and that only BEs may stall LEF extrusion, while it has been shown that other non-BE elements such as RNA polymerases could also act as partial extrusion barriers [5,8,9].”

“In the bacterial and mammalian systems, RNA polymerase II functions as a moving barrier for loop extrusion (33, 43).”

“Together with the recent report on the interplay between elongating RNA polymerases (RNAPs) and condensin (44), these results hint that the evolution of the gene’s high expression is partly through the directional convergence between”

“The bulky transcriptional machinery is likely to influence the position of cohesinmediated DNA loops [96, 168] (Fig.”

“A similar interplay between transcription and loop extrusion has been observed in Bacteria and WAPL/CTCF-depleted mammalian cells, where loop extrusion is impeded by polymerases, while SMC complexes might be pushed by transcribing complexes 19,51 – 53 .”

“Furthermore, large protein complexes such as RNA polymerases are able to limit loop extrusion by SMC protein complexes, such as bacterial condensins and eukaryotic cohesins (Brandão et al., 2019; Hsieh et al., 2020; Krietenstein et al., 2020).”

“To test our predictions, we construct a simulation scheme for loop-extrusion on a replicating bacterial chromosome, by adapting a previously published algorithm for a single bacterial chromosome [32, 33], with system parameters modelled after C.”

“Recentstudies, both experimentalandtheoretical, have proposedthat the loop extrusion mechanism, which plays a crucial role in the formation of TADs, might have an impact on the transcription machinery 23,38,46,47,54 .”

“The depletion of Esa1 suppresses transcription thus reduces chromosome bound RNA polymerases, which may work as moving barriers (102).”

“On one hand, high concentrations of transcribing RNA Pol II have been proposed to act as permeable barriers that slow condensin loop extrusion (Brandão et al., 2019; Tran et al., 2017).”

“RNA polymerase is a known impediment for Smc DNA translocation (Brandão et al., 2019).”

“316 In this model highly transcribed genes might form a barrier to loop extrusion (66), due to 317 accumulation of large amounts of transcriptional machinery and regulatory factors.”

“Does loop extrusion work in concert with diffusion based mechanisms for distal VH genes, whereby stalling of the loop acts to tether a number of genes in close proximity to the recombination center, obviating the need for juxtaposed CBEs? It is also feasible that other loop impeding mechanisms, such as transcription (…”

“…histones H3 and H4 on chromatin ( Shintomi et al., 2015 ), we used mouse sperm, which have previously been shown to condense in a condensin-dependent manner in Xenopus egg extracts ( Shintomi et al., 2017 ) but which contain very low levels of histones H3 and H4 ( Brykczynska et al., 2010 ).”

“Despite such challenges, bottom-up models can provide conceptual insight into how various molecular mechanism control bacterial chromosome organization.”

“, 2004), possibly by the action of RNA polymerases (Heinz et al., 2018; Brandão et al., 2019).”

“Along this line, recent experiments showed that transcription may influence cohesin localization [108,109] or translocation [77, 103] but may also act as moving barriers against condensin progression [104].”

“…protein,which loadsbsuSmc complexes ontothe chromosome at parS sites near the replication origin. bsuSmc loading at parS sites aligns their translocation with replication forks and most transcription units, minimizing the risk of head-on collisions 33,34 .”

“[92,93] Specifically, RNA polymerases can act as moving barriers for cohesin, and cohesin velocity depends on the direction of gene transcription.”

“One prediction of the permeable moving barrier model is that the orientation of transcription impacts the distribution pattern of condensin (Brandão et al, 2019).”

“The reduction of Esa1-mediated histone acetylation may reduce chromosome-bound RNA polymerases, which may increase cohesin movement speed along chromatin to generate larger chromatin loops and shorter axes [117].”

“For instance, RNA polymerases have been reported to stall SMC complexes at highly transcribed genes, perhaps as a consequence of DNA coverage by so-called polymerase trains 32,38–41 .”

“Candidate barriers for these loops are G-rich sequences frequently found at promoters capable of forming G-quadruplex (G4) structures when not covered with nucleosomes, transcription termination sites (TTS) of actively transcribed genes, and RNAPII at the TTS. are less stable and have significantly less cohesin based…”

“Large proteins such as RNAPolII may also interfere with loop extrusion [65].”

“…how loop extrusion impacts chromatin folding and the global chromatin structure, examining the effects of different types of LE (one-sided, two-sided, symmetrical, asymmetrical) [16, 18, 20] and exploring the influence of other protein remodelers, such as RNApII, on cohesin dynamics [21–24].”

“Rather, by analogy with the accumulation of condensin at highly expressed genes ( Brandão et al., 2019 ; Rivosecchi et al., 2021 ), we speculate that arrays of Taz1 proteins tightly bound to telomere repeats might create a permeable barrier onto which condensin molecules accumulate.”

“…multiple consequences for DNA transactions, including transcription, DNA repair, and segregation, as well as contribute to the regulation of the extrusion of large DNA loops by bacteria condensins as they travel along the chromosome (Mäkelä and Sherratt 2020; Wang et al. 2017; Brandão et al. 2019).”

“…DC) loaded at rex sites on the X chromosome in XX hermaphrodites C. elegans animals (Morao et al, 2022), which enabled studying the loop extrusion activities of these SMCs and identify roadblocks in vivo with Hi-C (Morao et al, 2022; Brandão et al, 2019, 2021; Wang et al, 2017; Tran et al, 2017).”

“…loaded at rex sites on the X chromosome in XX hermaphrodites C. elegans animals (Morao et al , 2022), which enabled studying the loop extrusion activities of these SMCs and identify roadblocks in vivo with Hi-C (Morao et al , 2022; Brandão et al , 2021, 2019; Wang et al , 2017; Tran et al , 2017).”

“…it is essential to reveal what happens from a molecular point of view when the SMC complexes encounter obstacles on DNA, such as RNA polymerase (Brandão et al., 2019; Pradhan et al., 2022), chromatin proteins (Pradhan et al., 2022; Yamaura et al., 2024), transcription factors, and other SMC…”

“In the absence of DNA replication, SMCs translocating from the terminus to the origin still move against these dense RNA polymerases and might be slowed down by them 29,37,39,44 .”

“Recent studies have suggested that the transcriptional activity of RNA polymerase might be the driver behind the cohesin ring displacement yielding loop extrusion (Brandão et al., 2019; Lengronne et al., 2004).”

“Whilst this model can elegantly explain the bypassing of other SMCs [ 28 , 37 , 38 ] and large road-blocks [ 39 , 40 ], it cannot explain directed motion.”

“Broad cohesin ChIP-seq peaks are also thought to result from elongating RNAPII, which acts as a “moving barrier” to LE [93–95].”

“However, studies in bacteria showed that condensins can bypass the transcriptional machinery despite a reduction of speed upon their encounter (Brandao et al , 2019).”

“Two-sidedloopextrusionwasimplementedbycoupling1D stochasticmodelofcohesinkineticsto3Dpolymerdynamics,whereeachcohesin was represented as an additional harmonic bond between two beads (5, 79).”

“…leads to a global reduction in transcription levels ( Ž umer et al, 2024) and that the process of transcription in fl uences 3D genome organization (Banigan et al, 2023; Barshad et al, 2023; Brandão et al, 2019; Busslinger et al, 2017; Hsieh et al, 2020; Zhang et al, 2023; Zhang et al, 2021).”

“Previous studies have demonstrated that RNA polymerase can act as a "moving barrier" to extruding complexes in bacteria (35), yeast (36), slime mold Dictyostelium discoideum (38) and even for vertebrate cohesin (37).”

“Such insulation is believed to result from ability of transcribing RNA Pol II to slow-down cohesin and even push it back, known as “the moving barrier" mechanism seen both in mammals and in bacteria (Banigan et al., 2023; Brandão et al., 2019).”

“In bacteria, structural-maintenance-of-chromosome (SMC) proteins ( 51 ,52 ) and nucleoid associated proteins ( 53 ) also play a major role in forming DNA loops in vivo ( 54 ) (30–420 kb).”

“Recent studies suggest that genes themselves can serve as insulating elements [ 36 , 37 ], even without loop extrusion in yeast [ 38 ].”