Cairns Lab - Chromatin Modification Dynamics in Development

Another major area of interest in our laboratory is how chromatin remodeling/modification and key transcription factors help guide development of:

• germline stem cells,
• gametogenesis (sperm and egg),
• and early embryo development.

Our work in this area began with the first genome-wide examination of histone retention and histone modifications in the germline—focusing first in human sperm (Hammoud et al., Nature 2009). Remarkably, we found that genes of developmental importance are packaged/poised in a distinctive manner in the germline (sperm), a packaging that is likely to promote their proper regulation in the embryo.

How Chromatin and Transcription Factors Coordinate ZGA and Early Embryo Development—Using Zebrafish Germline and Early Embryos

We study zebrafish to address many mechanistic questions that are much more difficult to address in humans. Lab members are using the zebrafish model system to study the dynamic chromatin structure germ cells (sperm and eggs), fertilized zygotes, and early embryos—and the dynamic interplay of chromatin structure with transcription factors.

Zygotic genome activation (ZGA) is a fascinating developmental period: parental genomes largely harmonize their chromatin states (imprinted genes excepted), begin nascent transcription at proper genes, poise developmental genes for future activation, and repress most retroelements and repeat regions—to both achieve full developmental totipotency and protect the future germline. By leveraging the unique advantages provided by zebrafish, mouse and human systems, we aim to provide a deep mechanistic understanding of how transcription factors and chromatin packaging/marking work together to achieve developmental totipotency, properly execute ZGA, and help ensure proper initial developmental decision making. We have made major progress in three areas: ‘Placeholder’ nucleosomes, Establishment of Higher Order Chromatin in Zebrafish Germline and Early Embryos, and Dux Factors in Early Embryos and FSHD pathogenesis (see later section).

Placeholder Nucleosomes

We published previously that most developmental gene promoters in human and zebrafish sperm are packaged in bivalent chromatin (bearing H3K4me3 and H3K27me3) that lack DNAme, suggesting a ‘poised’ chromatin state (similar to ES cells) that might be inherited (Hammoud et al., Nature 2009). However, as early cleavage embryos were later shown to have very low/absent H3K27me3, germline bivalency is evidently not continuously maintained, questioning its purpose. To reconcile/address, our recent evidence supports instead a ‘handoff-handback’ model involving a ‘placeholder’ type of chromatin that is installed during early (pre-ZGA) embryos—upon which developmental gene bivalency is restored during post-ZGA (Murphy et al., Cell 2018). Specifically, we revealed the focal installation of special ‘Placeholder’ nucleosomes bearing histone variant H2A.Z (FV) and H3K4me1 into the same developmental loci in both the paternal and maternal genomes during preZGA. These two modifications/attributes deter DNAme, and reside at all the locations where bivalency will be re-established after ZGA. Placeholder nucleosomes are installed by the SRCAP remodeler, likely focally guided by transcription factors, and pruned by the chaperone ANP32E (Murphy et al., Cell 2018).

Recently, we greatly advanced this model by determining that PRC1 adds H2Aub to Placeholder nucleosomes at developmental genes prior to ZGA, which helps silence them during ZGA/post-ZGA stages. Furthermore, during post-ZGA the non-canonical PRC2 complex (bearing the H2Aub ‘reader’ Aebp2) then adds H3K27me3 (Hickey et al., BioRxiv 2021). Placeholder installation into the maternal genome during cleavage also explains the mechanistic basis for a second remarkable observation in zebrafish embryo epigenetics—that oocyte/maternal DNAme patterns are reprogrammed in cleavage-stage embryos to match the paternal pattern, even in the absence of the paternal genome (Potok et al., Cell, 2013). Finally, our recent work on 3D chromatin architecture has revealed the unique chromatin packaging of sperm chromatin in zebrafish, and shown that higher order chromatin first forms in embryos after ZGA at enhancer sites marked with high H3K27ac (Wike et al., Genome Research 2021). Taken together, our work explains several major steps in preZGA chromatin reprogramming and developmental gene marking and regulation in zebrafish.

Mouse & Human Germline Stem Cells and Spermatogenesis

Our lab is fascinated by germline stem cells, and how their development is regulated by the testis niche. Gametogenesis—the generation of mature sperm from differentiating spermatogonia—involves major changes in genome packaging, and results in the packaging of developmental genes in a ‘poised’ format that may promote their proper expression after fertilization, in the embryo and beyond.

Here, understanding male spermotogonial stem cell (SSC) development and differentiation are of fundamental interest to basic science and medicine/fertility. During the past five years, we applied single cell genomics, IHC validation and other approaches to our nationally-unique rapid autopsy testis collection to broadly pioneer the developmental progression of human SSCs, and their interactions with the somatic testis niche (seminiferous tubule) during prenatal, postnatal, pubescent, young adult and older adult stages. In 2017, we conducted the first single-cell analysis of human SSCs (Guo et al., Cell Stem Cell, 2017), defining four SSC ‘States’, and showed their sequential progression from quiescence (State 1), to proliferation (State 2), to metabolic upregulation (State 3), to commitment to differentiation/meiosis (State 4). We then conducted single-cell analysis of the entire adult testis to provide an ‘atlas’ of germline and somatic niche cell states, and gametogenesis. Importantly, we identified the long sought, most undifferentiated and quiescent postnatal germline SSC—which we termed ‘State 0’ (Guo et al., Cell Research, 2018). We then characterized human puberty, showing the derivation of Leydig and myoid cells from a common somatic precursor, the co-development of germline stem cells with niche development—and roles for testosterone by examining testes donated by testosterone-suppressed transgender females (Guo et al., Cell Stem Cell, 2020). We next examined (via collaboration) the embryonic and prenatal testis, and revealed that Sertoli and Leydig cells originate during fetal stages (week 6-7) from a common heterogeneous progenitor, and that male primordial germ cells transition (~week 16) into State 0-like spermatogonia (Guo et al., Cell Stem Cell, 2021). Notably, our submitted work (below) has compared young (~20yo) to older adults (>60yo), revealing new information about how spermatogonia and somatic cell niche change and dysregulate during aging (Guo et al., in review). Taken together, our series of papers has provided major new insights into SSCs and testis development, alongside foundational datasets for comparisons to infertility, cancer and aging.

We have also continuing interests in how DNA methylation changes in the germline, especially with aging and in cancer, and have worked in collaboration with Dr. Doug Carrell’s lab here at the University of Utah—who has special collections of sperm donors (Jenkins et al., PLoS Genetics, 2014)

Developmental Decision Making in the Early Mammalian Embryo

A major emerging interest in the lab is how the human embryo employs a collaboration between transcription and chromatin factors to achieve totipotency in mammalian embryos—the ability to become any embryonic or extra-embryonic cell type.

DUX Factors in Early Embryos

To identify transcriptional drivers of mammalian ZGA, we created high-resolution stranded transcriptomes of early human embryos. Our analyses revealed key roles for the double-homeodomain transcription factor hDUX4 (and its mouse ortholog, mDux) in activating ZGA genes and retroviral elements (Hendrickson et al., Nature Genetics 2017). Notably, mDux expression efficiently converted mouse embryonic stem cells (mESCs) into 2-cell-embryo-like ('2C-like') cells, greatly reduced POU5F1/OCT4 protein, dissolved chromocenters, and converted ESC chromatin to a state strongly resembling mouse 2C embryos (Hendrickson et al., Nature Genetics 2017). Although not essential for embryogenesis or ZGA, loss of mDux leads produces litters with half the normal survivors, defects in the progression to blastocyst stage, and lowers transcription of many ZGA genes. These results support roles in early embryo progression and function—likely with additional factors which we aim to characterize.

DUX is activated by p53 signaling in embryonic and FSHD cell models

mDux and hDUX4 are among the first genes transcribed at ZGA, and both reside in complex repeat arrays. Importantly, improper DUX4 activation/expression underlies Fascioscapulohumoral Muscular Dystophy (FSHD). Therefore, we sought a possible unifying mDux/hDUX4 gene activation strategy in embryos and in FSHD. Surprisingly, we discovered that Dux and DUX4 are both activated by p53 (Grow et al., Nature Genetics, 2021). p53 is activated in early cleavage embryos and in FSHD cells, and long-read sequencing and assembly of the mouse Dux repeat locus revealed its complex chromatin regulation, and both positive and negative feedback loops. Notably, Dux expression promoted the differentiation of ESCs into extra-embryonic cell types in embyroid body assays. Furthermore, iPS cells derived from patients with FSHD activated human DUX4 during p53 signaling via a p53-binding site we identified in a primate-specific subtelomeric long terminal repeat LTR10C element. Thus, p53 activation convergently evolved to couple p53 to Dux/DUX4 activation—potentially uniting the developmental and disease regulation of DUX-family factors and identifying evidence-based therapeutic opportunities for FSHD (Grow et al., Nature Genetics, 2021).