Chromosome Research

BackgroundWhile CENP-A is the epigenetic determinant of the centromeric function, the role of CENP-B, the sole centromeric protein binding a specific DNA sequence (CENP-B-box), remains elusive. In the few mammalian species analyzed so far, the CENP-B box is contained in the major satellite repeat that is present at all centromeres. We previously demonstrated that, in the genus Equus, some centromeres lack any satellite repeat. ResultsHere, we show that, in four Equus species, CENP-B is expressed but does not bind the numerous satellite-free and the majority of satellite-based centromeres while it is localized at several ancestral now inactive centromeres. The absence of CENP-B is related to the lack of CENP-B boxes rather than to peculiar features of the protein itself. CENP-B boxes are comprised in a previously undescribed repeat which is not the major satellite bound by CENP-A. Comparative sequence analysis suggests that this satellite was centromeric in the equid ancestor, lost centromeric function during evolution and gave rise to a short CENP-A bound repeat not containing the CENP-B box but being enriched in dyad symmetries. Centromeres lacking CENP-B are functional and recruit normal amounts of the centromeric proteins CENP-A and CENP-C. ConclusionsWe propose that the uncoupling between CENP-B and CENP-A may have played a role in the evolutionary reshuffling of equid centromeres. This study provides new insights into the complexity of centromere organization in a largely biodiverse world where the majority of mammalian species still have to be studied.

BackgroundOrganization of the eukaryotic genome is essential for proper function, including gene expression. In metazoans, chromatin loops and Topologically Associated Domains (TADs) organize genes into transcription factories, while chromosomes occupy nuclear territories in which silent heterochromatin is compartmentalized at the nuclear periphery and active euchromatin localizes to the nucleus center. A similar hierarchical organization occurs in the fungus Neurospora crassa where its seven chromosomes form a Rabl conformation typified by heterochromatic centromeres and telomeres independently clustering at the nuclear membrane, while interspersed heterochromatic loci aggregate across Megabases of linear genomic distance to loop chromatin in TAD-like structures. However, the role of individual heterochromatic loci in normal genome organization and function is unknown. ResultsWe examined the genome organization of a Neurospora strain harboring a [~]47.4 kilobase deletion within a temporarily silent, facultative heterochromatic region, as well as the genome organization of a strain deleted of a 110.6 kilobase permanently silent constitutive heterochromatic region. While the facultative heterochromatin deletion minimally effects local chromatin structure or telomere clustering, the constitutive heterochromatin deletion alters local chromatin structure, the predicted three-dimensional chromosome conformation, and the expression of some genes, which are qualitatively repositioned into the nucleus center, while increasing Hi-C variability. ConclusionsOur work elucidates how an individual constitutive heterochromatic region impacts genome organization and function. Specifically, one silent region indirectly assists in the hierarchical folding of the entire Neurospora genome by aggregating into the "typical" heterochromatin bundle normally observed in wild type nuclei, which may promote normal gene expression by positioning euchromatin in the nucleus center.

The three-dimensional configuration of the eukaryotic genome is an emerging area of research. Chromosome conformation capture outlined genome segregation into large scale A and B compartments corresponding mainly to transcriptionally active and repressive chromatin. It remains unknown how the compartmentalization of the genome changes in growing oocytes of animals with hypertranscriptional type of oogenesis. In this type of oogenesis, highly elongated chromosomes, called lampbrush chromosomes, acquire a characteristic chromomere-loop appearance, representing one of the classical model systems for studying the structural and functional organization of chromatin domains. Here, we compared the distribution of A/B compartments in chicken somatic cells with chromatin domains in lampbrush chromosomes. We found that in lampbrush chromosomes, the extended chromatin domains, restricted by compartment boundaries in somatic cells, disintegrate into individual chromomeres. Next, we performed FISH-mapping of the genomic loci, which belong to A or B chromatin compartments as well as to A/B compartment transition regions in embryonic fibroblasts on isolated lampbrush chromosomes. We established, that in chicken lampbrush chromosomes, clusters of dense compact chromomeres bearing short lateral loops and enriched with repressive epigenetic modifications generally correspond to constitutive B compartments in somatic cells. These results suggest that gene-poor regions tend to be packed into chromomeres. Clusters of small loose chromomeres with relatively long lateral loops show no obvious correspondence with either A or B compartment identity. Some genes belonging to facultative B (sub-) compartments can be tissue-specifically transcribed during oogenesis, forming distinct lateral loops.

The architecture of mammalian mitotic chromosomes is considered to be universal across species and cell types. However, some studies suggest that features of mitotic chromosomes might be cell type or species specific. We previously reported that CTCF binding in human differentiated cell lines is lost in mitosis, whereas mouse embryonic stem cells (mESC) display prominent binding at a subset of CTCF sites in mitosis. Here, we perform parallel footprint ATAC-seq data analyses of mESCs and somatic mouse and human cells to further explore these differences. We then investigate roles of mitotically bound (bookmarked) CTCF in prometaphase chromosome organization by Hi-C. We do not find any remaining interphase structures such as TADs or CTCF loops at mitotically bookmarked CTCF sites in mESCs. This suggests that mitotic loop extruders condensin I and II are not blocked by bound CTCF, and thus that any remaining CTCF binding does not alter mitotic chromosome folding. Lastly, we compare mitotic Hi-C data generated in this study in mouse with publicly available data from human and chicken cell lines. We do not find any cell type specific differences; however, we do find a difference between species. The average genomic size of mitotic loops is much smaller in chicken (200-350 kb), compared to human (500-750 kb) and mouse (1-2 mb). Interestingly, we find that this difference in loop size is correlated with the average genomic length of the q-arm in these species, a finding we confirm by microscopy measurements of chromosome compaction. This suggests that the dimensions of mitotic chromosomes can be modulated through control of sizes of loops generated by condensins to facilitate species-appropriate shortening of chromosome arms.

Karyotypes of less than 10% of bird species are known. Using immunolocalization of the synaptonemal complex, the core structure of meiotic chromosomes at the pachytene stage, and centromere proteins we described male pachytene karyotypes of seventeen species of birds. This method enables higher resolution than the conventional analyses of metaphase chromosomes. We provided the first descriptions of the karyotypes of three species (Rook, Blyths reed warbler and European pied flycatcher), corrected the published data on the karyotypes of ten species and confirmed them for four species. All passerine species examined have highly conservative karyotypes, 2n=80-82 with seven pairs of macrochromosomes and 33-34 pairs of microchromosomes. In all of them but not in the Common cuckoo we revealed single copies of the germline restricted chromosomes varying in size and morphology even between closely related species. This indicates a fast evolution of this additional chromosome. The interspecies differences concern the sizes of the macrochromosomes, morphology of the microchromosomes and sizes of the centromeres. The pachytene cells of the Gouldian finch, Brambling and Common linnet contained heteromorphic synaptonemal complexes indicating heterozygosity for inversions or centromere shifts. The European pied flycatcher, Gouldian finch and Domestic canary have extended centromeres in several macro- and microchromosomes.

Chromosome segregation in eukaryotes requires the orchestrated interaction of chromosomes with microtubules, mediated by the kinetochore multiprotein complex that assembles on specific chromosomal regions known as centromeres. In most eukaryotes, two centromeric proteins, CenH3 and Cenp-C, are essential for centromere function. In Drosophila, the localization of CenH3 (referred to as Cid in Drosophila) depends on its chaperone CAL1 and Cenp-C. Previous studies have shown that both Cid and Cenp-C underwent a coincident gene duplication and likely functional specialization in the Drosophila subgenus. Independently, Cid duplications led to Cid1, Cid3, and Cid4 paralogs in the montium group (Sophophora subgenus), but it is unknown whether this group also underwent parallel duplications of Cenp-C. Here, we investigate this possibility by analyzing sequenced genomes of 23 montium group species. We identified Cenp-C genes in five distinct syntenic loci; we named these genes Cenp-C1b, Cenp-C1c, Cenp-C1d, Cenp-C1e and Cenp-C3. Despite their distinct synteny, most montium group species only encode a single Cenp-C; their phylogeny mirrors the species phylogeny, and they appear to have retained Cenp-C protein motifs indicative of function. A closer examination revealed that these Cenp-C genes resulted from gene translocations or alternate retention (duplication followed by loss of the ancestral copy); only one species, D. vulcana, retains two intact Cenp-C paralogs. Therefore, unlike the Drosophila genus, the co-retention of three Cid paralogs in the montium group has not resulted in a coincident Cenp-C paralog co-retention. Our work highlights differences in functional retention and likely specialization of the two most conserved centromeric proteins in eukaryotes.

Advances in DNA sequencing technologies have, for the first time, provided us with enough whole chromosome-level genomes to understand in detail how chromosome number and composition change over time. Here, we use the genomes of butterflies and moths to look at the levels and age of macrosynteny in the Lepidoptera and Trichoptera. We used comparative BUSCO analsysis to define reproducible units of macrosynteny which we term Lepidopteran Synteny Units or LSUs. The 31 chromosomes of the model butterfly Melitaea cinxia served as a reference point. The results show that chromosome-wide macrosynteny extends from the most basal branches of the Lepidopteran phylogeny to the most distal. This synteny also extends to the order Trichoptera, a sister group of the Lepidoptera. Thus, chromosome-wide macrosynteny has been conserved for a period of >200 My in this group of insects. We found no major interchromosomal translocations, reciprocal or non-reciprocal, in the genomes studied. Intrachromosomal rearrangements, in contrast, were abundant. Beyond its use in defining LSUs, this type of homology-based analysis will be useful in determining the relationships between chromosomal elements in different animals and plants. Further, by more precisely defining the breakpoints of chromosomal rearrangements we can begin to look at their potential roles in chromosomal evolution. StatementThe authors declare no conflicting interests ContributionsConceptualisation: W.T., R.H.f.; data analysis: W.T.; writing & editing: W.T., K.S., R.H.f All authors read and approved the final manuscript.

Reptiles exhibit a variety of modes of sex determination, including both temperature-dependent and genetic mechanisms. Among those species with genetic sex determination, sex chromosomes of varying heterogamety (XX/XY and ZZ/ZW) have been observed with different degrees of differentiation. Karyotype studies have demonstrated that Gila monsters (Heloderma suspectum) have ZZ/ZW sex determination and this system is likely homologous to the ZZ/ZW system in the Komodo dragon (Varanus komodoensis), but little else is known about their sex chromosomes. Here, we report the assembly and analysis of the Gila monster genome. We generated a de novo draft genome assembly for a male using 10X Genomics technology. We further generated and analyzed short-read whole genome sequencing and whole transcriptome sequencing data for three males and three females. By comparing female and male genomic data, we identified four putative Z-chromosome scaffolds. These putative Z-chromosome scaffolds are homologous to Z-linked scaffolds identified in the Komodo dragon. Further, by analyzing RNAseq data, we observed evidence of incomplete dosage compensation between the Gila monster Z chromosome and autosomes and a lack of balance in Z-linked expression between the sexes. In particular, we observe lower expression of the Z in females (ZW) than males (ZZ) on a global basis, though we find evidence suggesting local gene-by-gene compensation. This pattern has been observed in most other ZZ/ZW systems studied to date and may represent a general pattern for female heterogamety in vertebrates.

A few species have evolved multiple sex chromosome systems with more than two Xs or Ys. These involve sex chromosome-autosome translocations (sometimes called fusions as very small heterochromatic arms may be deleted), creating neo-sex chromosome systems. Among vertebrates, frogs (Anura) have the highest known number of such translocation systems. This study within the genus Leptodactylus, investigated the two species L. pentadactylus (LPE) and L. paraensis (LPA), in which large ring multivalents are seen in male meiosis, indicating translocations involving the sex chromosomes. Four other species studied do not have such rings, but they share characteristics making rearrangements less likely to be eliminated. To start understanding the formation of multivalents, we used genomic and cytogenetic methods to investigate repetitive DNA sequences, including satellite DNAs, rDNAs, and telomeric sequences, and conducted comparative genomic hybridization (CGH). The LPE genome includes a large number of satDNA families, and in situ mapping of several satDNAs individually identified eight of the ten chromosomes in its multivalent. In LPA, morphological similarities indicate that several chromosomes are shared by the multivalents of both species, and a candidate ancestral sex chromosome pair could be identified. In situ mapping in LPE suggests recent satDNA accumulation in the subtelomeric regions, which differ from those in the outgroup species, contrary to the expectation that the translocations create sex-linkage in the pericentromeric regions.

Segregation of chromosomes depends on the centromere. Most species are monocentric, with the centromere restricted to a single region per chromosome. In some organisms, monocentric organization changed to holocentric, in which the centromere activity is distributed over the entire chromosome length. However, the causes and consequences of this transition are poorly understood. Here, we show that the transition in the genus Cuscuta was associated with dramatic changes in the kinetochore, a protein complex that mediates the attachment of chromosomes to microtubules. We found that in holocentric Cuscuta species the KNL2 genes were lost; the CENP-C, KNL1, and ZWINT1 genes were truncated; the centromeric localization of CENH3, CENP-C, KNL1, MIS12, and NDC80 proteins was disrupted; and the spindle assembly checkpoint (SAC) was degenerated. Our results demonstrate that holocentric Cuscuta species lost the ability to form a standard kinetochore and do not employ SAC to control the attachment of microtubules to chromosomes.

Chromatin immunoprecipitation (ChIP) assays provide quantitative information about the genomic localization of chromatin-binding proteins. However, their sensitivity is limited by several technical variables. To generate high-confidence datasets, in this protocol, we used the Saccharomyces cerevisiae chromatin as an exogenous spike-in control for the ChIP of two S. pombe heterochromatin-associated proteins. This permitted normalization of the ChIP signals based on immunoprecipitation efficiencies across samples. Here, we describe the steps for spike-in control preparation, validation, and its use in data normalization. For complete details on the use and execution of this protocol, please refer to Khanduja et al.1

BackgroundMaize knobs are regions of constitutive heterochromatin that are readily identified in both meiotic and somatic chromosomes. These structures have been characterized as stable throughout the cell cycle, exhibiting late replication during the S-phase, and are composed of two specific families of highly repetitive DNA sequences: K180 and TR-1. Although widely used as cytogenetic markers due to their variability in number and chromosomal position across inbred lines, hybrids, and landraces, little is known about their chromatin structure and dynamics. In this study, we analyzed chromatin accessibility of knobs using DNS-seq data across four maize tissues representing distinct developmental stages. ResultsOur results reveal that K180 knobs exhibit tissue-specific variation in chromatin accessibility, transitioning between open and closed states during development. In contrast, the TR-1 knob of chromosome 4 remained consistently inaccessible across all tissues analyzed. A knob composed of both K180, and TR-1 further supported this observation, with only the K180 region showing dynamic accessibility. To validate these findings, we also analyzed other repetitive regions such as centromeres, which showed a uniformly closed chromatin structure similar to TR-1. These results suggest a unique developmental modulation of chromatin accessibility associated with K180 repeats. While the chromatin accessibility of knobs does not reach the levels observed at Transcription Start Sites (TSS), the comparison among different classes of repetitive DNA within maize constitutive heterochromatin provides compelling evidence for sequence-specific and tissue-specific chromatin dynamics. ConclusionsOur findings uncover a previously unrecognized property of maize knobs and establish a reference for future studies on chromatin organization and epigenetic regulation of repetitive DNA in plant genomes.

We present a SNP-based crossover map for Drosophila mauritiana. Using females derived by crossing two different strains of D. mauritiana, we analyzed crossing over on all five major chromosome arms. Analysis of 105 male progeny allowed us to identify 327 crossover chromatids bearing single, double, or triple crossover events, representing 398 separate crossover events. We mapped these crossover sites along these five chromosome arms using a genome sequence map that includes the euchromatin-heterochromatin boundary. Confirming previous studies, we show that the overall crossover frequency in D. mauritiana is higher than is seen in D. melanogaster. Much of the increase in exchange frequency in D. mauritiana is due to a greatly diminished centromere effect. Using larval neuroblast metaphases from D. mauritiana -D. melanogaster hybrids we show that the lengths of the pericentromeric heterochromatin do not differ substantially between the two species, and thus cannot explain the observed differences in crossover distribution. Using a new and robust maximum likelihood estimation tool for obtaining Weinstein tetrad distributions, we observed an increase in bivalents with two or more crossovers when compared to D. melanogaster. This increase in crossing over along the arms of D. mauritiana likely reflects an expansion of the crossover-available euchromatin caused by the reduction in the centromere effect. The pattern of crossing over in D. mauritiana conflicts with the commonly accepted view of centromeres as polar suppressors of exchange (whose intensity is buffered by sequence non-specific heterochromatin) and demonstrates the importance of expanding such studies into other species of Drosophila. Article SummaryIn meiosis, homolog segregation is usually ensured by crossovers. The number and distribution of crossovers is in part regulated by cis-acting factors such as the cis- acting centromere effect, a polar suppression of exchange emanating from the vicinity of the centromere. We use SNP-based crossover mapping to show that in Drosophila mauritiana, the centromere effect is greatly reduced on four of the five major chromosome arms. We conclude that the centromere effect differs between Drosophila mauritiana and Drosophila melanogaster and that the ability to attenuate the centromere effect is not a general property of heterochromatin.

Although the spatial organization of the genome is closely linked to biological function, genome structure is highly stochastic. Within this heterogeneity, specific function-associated structural elements must be maintained, even when the nucleus is deformed due to physiological physical constraints. The radial positioning of genomic loci - their distance from the nuclear periphery - has long been considered an important feature of genome organization which is correlated with both structural elements and genomic activity. In the current study, we developed an experimental system for manipulating the radial positioning of the genome, by expressing the sperm-specific protein Prm1 in somatic cells. By microscopy, we observe that initial Prm1 nuclear foci develop within 72 hours into a large Prm1 focus occupying most of the nuclear interior, while the entire genome is driven towards the nuclear periphery, resulting in a 3-5 fold reduction in the volume that the genome occupies. Noting that this system enables isolation of a pure population of cells with reorganized nuclei, we then used Hi-C to study the effects of this perturbation. Remarkably, we find that interaction patterns are largely robust to this major nuclear reorganization, with minor changes which mostly reflect a strengthening of heterochromatin self-interactions. Our experimental system provides means for manipulating nuclear organization in a reproducible manner, potentially allowing to examine radial positioning decoupled from other features of genome organization. Highlighting the complementary nature of microscopic and genomic methods, our work further suggests a remarkable resilience of genome structure such that large-scale nuclear changes, including chromosome compression and changes in radial positioning, can occur without extensive alteration of functional genome organization.

As part of the Synthetic Yeast 2.0 (Sc2.0) project, we designed and synthesized synthetic chromosome I. The total length of synI is [~]21.4% shorter than wild-type chromosome I, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns of potential instability and karyotype imbalance. We used a variation of a previously developed, robust CRISPR-Cas9 method to fuse chromosome I to other chromosome arms of varying length: chrIXR (84kb), chrIIIR (202kb) and chrIVR (1Mb). All fusion chromosome strains grew like wild-type so we decided to attach synI to synIII. Through the investigation of three-dimensional structures of fusion chromosome strains, unexpected loops and twisted structures were formed in chrIII-I and chrIX-III-I fusion chromosomes, which depend on silencing protein Sir3. These results suggest a previously unappreciated 3D interaction between HMR and the adjacent telomere. We used these fusion chromosomes to show that axial element Red1 binding in meiosis is not strictly chromosome size dependent even though Red1 binding is enriched on the three smallest chromosomes in wild-type yeast, and we discovered an unexpected role for centromeres in Red1 binding patterns.

Drosophila miranda is considered an excellent model for studying sex chromosome evolution due to its neo-sex chromosomes, which originated from fusions between autosomes and sex chromosomes. In this study, we took advantage of the latest genome assembly of D. miranda to design the first oligo probe libraries targeting neo-sex chromosomes, covering X and Y-linked regions with times ranging from [~]1.5 to 60 million years. These libraries, which include both single-copy and repetitive oligos, were generated by integrating the OligoY approach to the conventional OligoMiner pipeline and validated through fluorescence in situ hybridization (FISH). We optimized oligo density and spacing parameters to predict consistent and effective chromosome painting. Beyond tool improvement, our mapping of the three largest unplaced Y-linked scaffolds in D. miranda reveals a complex evolutionary mechanism driving the current structure of the Y chromosome, including chromosomal translocation, centromere loss, and inversions. This work provides essential tools for sex chromosome identification via probe labeling and offers a foundation for exploring the spatial and evolutionary dynamics of sex chromosomes across different cell types. Author summaryWhile previous studies have focused on using single-copy oligonucleotides for chromosome painting, these oligos have limited effectiveness in targeting repetitive regions such as ribosomal DNA, pericentromeres, and mainly Y chromosomes. In this study, we integrated the OligoMiner and OligoY pipelines to design highly specific oligonucleotide libraries capable of targeting both single-copy and repetitive regions in any chromosome, enabling comprehensive painting of autosome and sex chromosomes. Using Drosophila miranda neo-sex chromosomes as a model, we validated the specificity of our oligo libraries through fluorescence in situ hybridization (FISH). Our results demonstrate that it is possible to achieve successful chromosome painting of sex chromosomes ranging from 1.5 to 60 million years old by combining single-copy and repetitive oligos, without compromising specificity. Notably, we painted the neo-Y chromosome of D. miranda and proposed a hypothesis to give rise to its current structure. This approach provides a powerful tool for studying chromosome evolution and organization, particularly in complex and repetitive genomic regions.

Chromosomal rearrangements are crucial in speciation, acting as barriers to gene flow. Holocentric chromosomes, such as those in Lepidoptera, can facilitate karyotype changes. Despite chromosome fusions being more common, speciation events are mostly linked to fissions. Notable karyotypic variation is observed in three clades of the subfamily Polyommatinae (Lycaenidae), with chromosome numbers ranging from n = 10 to n = 225. This study used flow cytometry and molecular cytogenetic analyses to investigate genome sizes and karyotypes in several species of the genera Polyommatus and Lysandra with derived and modal chromosome numbers. The findings show no support for polyploidy, supporting karyotypic diversification via fragmentation of chromosomes. Species with high chromosome numbers have larger genomes, which indicates a potential role of mobile elements but contradicts the hypothesis of holocentric drive. Telomeric signals were detected at the ends of fragmented chromosomes. No interstitial telomeric sequences were detected on autosomes. Interstitial telomeric signals on sex chromosomes, however, revealed multiple sex chromosome systems in Polyommatus dorylas and Polyommatus icarus, with two karyotype races differing in sex chromosome constitution in the latter. Pool-seq and coverage analyses indicated shared fusion of sex chromosomes with an autosome bearing the rDNA locus, followed by a fusion with chromosome 20 in the Czech population. Notably, the W chromosome resists fragmentation, likely due to epigenetic silencing protecting it from activity of mobile elements.

Flow cytometry estimates of genome sizes among species of Drosophila show a 3-fold variation, ranging from [~]127 Mb in Drosophila mercatorum to [~]400 Mb in Drosophila cyrtoloma. However, the assembled portion of the Muller F Element (orthologous to the fourth chromosome in Drosophila melanogaster) shows a nearly 14-fold variation in size, ranging from [~]1.3 Mb to > 18 Mb. Here, we present chromosome-level long read genome assemblies for four Drosophila species with expanded F Elements ranging in size from 2.3 Mb to 20.5 Mb. Each Muller Element is present as a single scaffold in each assembly. These assemblies will enable new insights into the evolutionary causes and consequences of chromosome size expansion.

Single-stranded DNA gaps form within the E. coli chromosome during replication, repair and recombination. However, information about the extent of ssDNA creation in the genome is limited. To complement a recent whole-genome sequencing study revealing ssDNA gap genomic distribution, size, and frequency, we used fluorescence microscopy to monitor the spatiotemporal dynamics of single-stranded DNA within live E. coli cells. The ssDNA was marked by a functional fluorescent protein fusion of the SSB protein that replaces the wild type SSB. During log-phase growth the SSB fusion produces a mixture of punctate foci and diffuse fluorescence spread throughout the cytosol. Many foci are clustered. Fluorescent markers of DNA polymerase III frequently co-localize with SSB foci, often localizing to the outer edge of the large SSB features. Novel SSB-enriched features form and resolve regularly during normal growth. UV irradiation induces a rapid increase in SSB foci intensity and produces large features composed of multiple partially overlapping foci. The results provide a critical baseline for further exploration of ssDNA generation during DNA metabolism. Alterations in the patterns seen in a mutant lacking RecB function tentatively suggest associations of particular SSB features with the repair of double strand breaks and post-replication gaps.

Replication Timing (RT) refers to the temporal order in which the genome is replicated during S phase. Early replicating regions correlate with the transcriptionally active, accessible euchromatin (A) compartment, while late replicating regions correlate with the heterochromatin (B) compartment and repressive histone marks. Previously, widespread A/B genome compartmentalization changes were reported following Brd2 depletion. Since RT and A/B compartmentalization are two of the most highly correlated chromosome properties, we evaluated the effects of Brd2 depletion on RT. We performed E/L Repli-Seq following Brd2 depletion in the previously described Brd2 conditional degron cell line and found no significant alterations in RT after Brd2 KD. This finding prompted us to re-analyze the Micro-C data from the previous publication. We report that we were unable to detect any compartmentalization changes in Brd2 depleted cells compared to DMSO control using the same data. Taken together, our findings demonstrate that Brd2 depletion alone does not affect A/B compartmentalization or RT in mouse embryonic stem cells.