Variation and Evolution of Human Centromeres: A Field Guide and Perspective

We are entering a new era in genomics where entire centromeric regions are accurately represented in human reference assemblies. Access to these high-resolution maps will enable new surveys of sequence and epigenetic variation in the population and offer new insight into satellite array genomics and centromere function. Here, we focus on the sequence organization and evolution of alpha satellites, which are credited as the genetic and genomic definition of human centromeres due to their interaction with inner kinetochore proteins and their importance in the development of human artificial chromosome assays. We provide an overview of alpha satellite repeat structure and array organization in the context of these high-quality reference data sets; discuss the emergence of variation-based surveys; and provide perspective on the role of this new source of genetic and epigenetic variation in the context of chromosome biology, genome instability, and human disease.

Isolated Changes in Chromatin Accessibility and Enhancer-Promoter Contacts at the β-Globin Locus Distinguish Fetal Hemoglobin Producing F-Cells from a-Cells

Reversal of the developmental switch from fetal (HbF, α 2γ 2) to adult (HbA,α 2β 2) hemoglobin is an important therapeutic approach for sickle cell disease (SCD) and β-thalassemia. It has been noted since the 1950s that a small number of circulating red blood cells, called F-cells, produce elevated levels of HbF; these cells are resistant to sickling and are present in increased numbers in patients with SCD and those treated with pharmacological HbF inducers such as hydroxyurea. Because successful therapy for SCD requires increasing the number of F-cells, it is imperative to understand how these cells arise. This can potentially occur through a shift towards a global fetal-like program, selective variation in levels of known HbF silencers such as BCL11A or LRF, or through discrete epigenetic changes at the β-globin locus. We previously began to address this clinically important question using a novel experimental approach of sorting cultured primary human erythroblasts into HbF-high (F-cell) and HbF-low (A-cell) populations (Khandros et al, Blood 2020). We showed that surprisingly, F-cells from healthy donor primary erythroid cultures have minimal transcriptional differences with A-cells. Unexpectedly, this was also the case when comparing responders (F-cells) and non-responders (A-cells) to treatment with the HbF inducers pomalidomide and hydroxyurea, and there were no differences in the expression of known HbF regulators. We therefore hypothesize that HbF synthesis in F-cells is determined by epigenetic variation confined to the β-globin locus (and not by global changes in the cell fate or nuclear milieu). To test this hypothesis, we compared genome wide chromatin accessibility by Assay for Transposase-Accessible Chromatin (ATAC-seq) in differentiation stage-matched F- and A-cells from healthy donor primary erythroid cultures, treated with vehicle, hydroxyurea, or pomalidomide. We observed striking similarities between F- and A-cells: out of 83,295 peaks called across all conditions, a mere five regions of differential accessibility were found, all at the β-globin locus (at the promoters and 3' UTR regions of the HBG1 and HBG2 genes as well as the BGLT3 non-coding RNA and HBBP1 pseudogene). This remarkable similarity in the global chromatin landscape between A- and F-cells cements the notion that these cells are fundamentally the same in terms of developmental and differentiation states, and that local epigenetic variation at the β-globin locus underlies the differences in HbF production. We also found that the gains in ATAC signal at the HBG1/2 genes were the most pronounced in F-cells from pomalidomide treated cultures, consistent with our finding that F-cells that arise following pomalidomide treatment have a higher content of HBG1/2 transcripts per cell. Drug treatments led to a larger number of changes in ATAC-seq peaks, at 123 and 1015 sites for treatment with hydroxyurea or pomalidomide, respectively, compared to vehicle. However, since differences at only 5 ATAC-seq peaks were observed between between F- and A-cells, we infer that the broader changes upon drug treatment are not needed for the phenotypic differences between F- and A-cells. Since transcription of the β-type globin genes is controlled by developmental stage-specific long-range contacts between the gene promoters and the locus control region (LCR), we determined whether the increase chromatin accessibility at the γ-globin genes in F-cells was associated with enhanced contacts with the LCR. Capture-C experiments revealed increased LCR-HBG1/2 promoter contacts and reduced LCR contacts with the adult HBB and HBD promoters in F-cells vs A-cells, demonstrating that local gains in chromatin accessibility are linked to long-range enhancer promoter contacts. Additionally, we did not detect differences in long-range chromatin contacts at several developmentally regulated genes, including LIN28B and BCL11A, solidifying the idea that γ-globin production in F-cells is specified locally through chromatin accessibility and chromatin architecture. In sum, our studies demonstrate that in adults, F-cells do not arise through reversion to a fetal like state or variation in expression of any known HbF regulator. Rather these cells reflect highly localized, perhaps stochastic modulation of chromatin architecture at the β-globin locus. Disclosures Blobel: Fulcrum Therapeutics, Inc.: Consultancy; Pfizer: Consultancy.

Complementary genomic and epigenomic adaptation to environmental heterogeneity

While adaptation is commonly thought to result from selection on DNA sequence-based variation, recent studies have highlighted an analogous epigenetic component as well. However, the extent to which these adaptive mechanisms to adaptation to environmental heterogeneity are redundant or complementary remains unclear. To address the underlying genetic and epigenetic mechanisms and their relationship underlying environmental adaptation, we screened the genomes and epigenomes of nine global populations of a predominately sessile marine invasive tunicate, Botryllus schlosseri. We detected clear population genetic and epigenetic differentiation, which were both significantly influenced by local environments, and the minimum annual sea surface temperature (T_min) was simultaneously identified as the top explanatory variable for both types of variation. However, there remain some degree of difference in population structure patterns between two levels, suggesting a certain level of autonomy in epigenetic variation. From the functional perspective, a set of functional genes and biological pathways were shared between two levels, indicating a conjoint contribution of genetic and epigenetic variation to environmental adaptation. Moreover, we also detected genetic- or epigenetic-specific genes/pathways in relation to a wide variety of core processes potentially underlying adaptation to local environmental factors, suggesting the partly independent relationship between two mechanisms. We infer that complementary genetic and epigenetic routes to adaptation are available in this system. Collectively, these mechanisms may facilitate population persistence under environmental changes and sustain successful invasions in novel but contrasting environments.

Phenotypic diversity created by a transposable element increases productivity and resistance to competitors in plant populations

An accumulating body of evidence indicates that natural plant populations harbour a large diversity of transposable elements (TEs). TEs provide genetic and epigenetic variation that can substantially translate into changes in plant phenotypes. Despite the wealth of data on the ecological and evolutionary effects of TEs on plant individuals, we have virtually no information on the role of TEs on populations and ecosystem functioning. On the example of Arabidopsis thaliana, we demonstrate that TE-generated variation creates differentiation in ecologically important functional traits. In particular, we show that Arabidopsis populations with increasing diversity of individuals differing in copy numbers of the ONSEN retrotransposon had higher phenotypic and functional diversity. Moreover, increased diversity enhanced population productivity and reduced performance of interspecific competitors. We conclude that TE-generated diversity can have similar effects on ecosystem as usually documented for other biological diversity effects.

Integrative analysis of epigenetics data identifies gene-specific regulatory elements

Understanding how epigenetic variation in non-coding regions is involved in distal gene-expression regulation is an important problem. Regulatory regions can be associated to genes using large-scale datasets of epigenetic and expression data. However, for regions of complex epigenomic signals and enhancers that regulate many genes, it is difficult to understand these associations. We present StitchIt, an approach to dissect epigenetic variation in a gene-specific manner for the detection of regulatory elements (REMs) without relying on peak calls in individual samples. StitchIt segments epigenetic signal tracks over many samples to generate the location and the target genes of a REM simultaneously. We show that this approach leads to a more accurate and refined REM detection compared to standard methods even on heterogeneous datasets, which are challenging to model. Also, StitchIt REMs are highly enriched in experimentally determined chromatin interactions and expression quantitative trait loci. We validated several newly predicted REMs using CRISPR-Cas9 experiments, thereby demonstrating the reliability of StitchIt. StitchIt is able to dissect regulation in superenhancers and predicts thousands of putative REMs that go unnoticed using peak-based approaches suggesting that a large part of the regulome might be uncharted water.

Leveraging the Mendelian disorders of the epigenetic machinery to systematically map functional epigenetic variation

Although each Mendelian Disorder of the Epigenetic Machinery (MDEM) has a different causative gene, there are shared disease manifestations. We hypothesize that this phenotypic convergence is a consequence of shared epigenetic alterations. To identify such shared alterations, we interrogate chromatin (ATAC-seq) and expression (RNA-seq) states in B cells from three MDEM mouse models (Kabuki [KS] type 1 and 2 and Rubinstein-Taybi type 1 [RT1] syndromes). We develop a new approach for the overlap analysis and find extensive overlap primarily localized in gene promoters. We show that disruption of chromatin accessibility at promoters often disrupts downstream gene expression, and identify 587 loci and 264 genes with shared disruption across all three MDEMs. Subtle expression alterations of multiple, IgA-relevant genes, collectively contribute to IgA deficiency in KS1 and RT1, but not in KS2. We propose that the joint study of MDEMs offers a principled approach for systematically mapping functional epigenetic variation in mammals.