The accessible chromatin landscape of the human genome

DNaseI hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers, and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ~2.9 million DHSs that encompass virtually all known experimentally-validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation, and regulatory factor occupancy patterns. We connect ~580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is choreographed with dozens to hundreds of co-activated elements, and the trans-cellular DNaseI sensitivity pattern at a given region can predict cell type-specific functional behaviors. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation

The accessible chromatin landscape of the human genome

DNaseI hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers, and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ~2.9 million DHSs that encompass virtually all known experimentally-validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation, and regulatory factor occupancy patterns. We connect ~580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is choreographed with dozens to hundreds of co-activated elements, and the trans-cellular DNaseI sensitivity pattern at a given region can predict cell type-specific functional behaviors. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation

Regulation of cell fate by the stem cell factors Cdx2, Oct4 and Sox2 in the early mouse embryo

Pluripotent cells have the potential to create any cell type, and therefore represent a source of healthy cells by which to replace diseased cells in regenerative therapies. Thus, understanding how pluripotent cells are established and regulated is a major milestone in developing safe and effective regenerative therapies. In the mammalian embryo pluripotent cells emerge through two rounds of cell fate decisions that in addition to establishing pluripotent cells, establish two differentiated cell lineages. The molecules and signaling pathways that regulate these cell fate decisions represent key players in the establishment and regulation of pluripotent cells. In this thesis, I use the mouse embryo as a system in which to gain insight into how pluripotency is established and regulated. Cdx2, Oct4 and Sox2 are transcription factors that are important regulators of pluripotency in vitro, but their role in regulating the cell fate decisions that establish pluripotency in vivo is less clear. By using a loss-of-function approach, I define the roles of each of these factors in regulating the cell fate decisions that establish pluripotency in the early mouse embryo. First, by eliminating maternal and zygotic Cdx2 I demonstrate that Cdx2 is not a maternal determinant for pluripotency or differentiation during early embryo cell fate decisions. Second, I eliminate both maternal and zygotic Oct4 and demonstrate that the initial specification of pluripotent and non-pluripotent lineages in the early mouse embryo is independent of Oct4, and that instead, Oct4 cell-autonomously regulates both formation of pluripotent cells, and differentiation of a non-pluripotent lineage, the primitive endoderm. Third, I demonstrate that in contrast to Oct4, Sox2 promotes differentiation of primitive endoderm non-cell autonomously. These results support the model that pluripotency is acquired through these early embryonic cell fate decisions, rather than being determined during early embryogenesis. Furthermore, analysis of the roles of Oct4 and Sox2 in these early embryonic cell fate decisions highlights distinct roles for these factors in establishing pluripotency and suggests that although in vitro Oct4 and Sox2 appear to have overlapping roles, these master regulators of pluripotency have distinct cell type specific roles in regulating the cell fate decisions that establish pluripotency in the early mouse embryo. Taken together, the research presented in this thesis greatly increases our understanding of the regulation of the cell fate decisions that establish pluripotency in the embryo and provides important insights into the biology of key regulators of pluripotency in vitro and in vivo

Understanding Human Lung Development through In Vitro Model Systems

An abundance of information about lung development in animal models exists; however, comparatively little is known about lung development in humans. Recent advances using primary human lung tissue combined with the use of human in vitro model systems, such as human pluripotent stem cell‐derived tissue, have led to a growing understanding of the mechanisms governing human lung development. They have illuminated key differences between animal models and humans, underscoring the need for continued advancements in modeling human lung development and utilizing human tissue. This review discusses the use of human tissue and the use of human in vitro model systems that have been leveraged to better understand key regulators of human lung development and that have identified uniquely human features of development. This review also examines the implementation and challenges of human model systems and discusses how they can be applied to address knowledge gaps.Human in vitro model systems have been instrumental to understand specific features of lung development that are unique to humans. In this review, how these models have been used to interrogate human lung development is explored, unlocking the potential to answer unknown questions in human biology and physiology.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155532/1/bies202000006_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155532/2/bies202000006.pd

HIPPO pathway members restrict SOX2 to the inner cell mass where it promotes ICM fates in the mouse blastocyst.

Pluripotent epiblast (EPI) cells, present in the inner cell mass (ICM) of the mouse blastocyst, are progenitors of both embryonic stem (ES) cells and the fetus. Discovering how pluripotency genes regulate cell fate decisions in the blastocyst provides a valuable way to understand how pluripotency is normally established. EPI cells are specified by two consecutive cell fate decisions. The first decision segregates ICM from trophectoderm (TE), an extraembryonic cell type. The second decision subdivides ICM into EPI and primitive endoderm (PE), another extraembryonic cell type. Here, we investigate the roles and regulation of the pluripotency gene Sox2 during blastocyst formation. First, we investigate the regulation of Sox2 patterning and show that SOX2 is restricted to ICM progenitors prior to blastocyst formation by members of the HIPPO pathway, independent of CDX2, the TE transcription factor that restricts Oct4 and Nanog to the ICM. Second, we investigate the requirement for Sox2 in cell fate specification during blastocyst formation. We show that neither maternal (M) nor zygotic (Z) Sox2 is required for blastocyst formation, nor for initial expression of the pluripotency genes Oct4 or Nanog in the ICM. Rather, Z Sox2 initially promotes development of the primitive endoderm (PE) non cell-autonomously via FGF4, and then later maintains expression of pluripotency genes in the ICM. The significance of these observations is that 1) ICM and TE genes are spatially patterned in parallel prior to blastocyst formation and 2) both the roles and regulation of Sox2 in the blastocyst are unique compared to other pluripotency factors such as Oct4 or Nanog

<i>Sox2</i> is restricted to EPI progenitors through an Fgf4/MEK-dependent mechanism.

<p>A) In wild-type (WT) embryos at early E3.75, NANOG is detected in a salt-and-pepper pattern in the ICM, while SOX2 begins to be downregulated in PE cells (arrowhead: cell in which NANOG is already downregulated, but SOX2 is not yet downregulated). B) In WT embryos at late E3.75, SOX2 and SOX17 are detected in a salt-and-pepper pattern in the ICM. C) At E4.25, SOX2 is exclusively detected in EPI and SOX17 in PE. D) At E3.75, SOX2 is detected in a larger proportion of ICM cells than is NANOG, indicating that NANOG is downregulated in the PE slightly before SOX2. E) FGF4/HEP is sufficient to repress SOX2 expression in the ICM since the SOX2-expressing proportion of ICM cells is reduced (and GATA6-expressing proportion concomitantly expanded) in wild-type embryos incubated in FGF4/HEP (avg. no. untreated ICM cells: 25.6+/−3.8; avg. no. treated ICM cells: 30.4+/−7.2). F) The downregulation of SOX2 in PE cells is dependent on FGFR/MEK, since the proportion of ICM cells expressing SOX2 is expanded (and the SOX17-expressing proportion concomitantly reduced) in wild-type embryos incubated in inhibitors of FGFR/MEK (avg. no. untreated ICM cells: 19.4+/−5.1; avg. no. treated ICM cells: 13+/−4.1). Bar  = 20 µm, p-value calculated by t-test.</p

Smad4 is essential for epiblast scaling and morphogenesis after implantation, but nonessential before implantation.

Peer reviewed: TrueAcknowledgements: We thank the lab of Dr David Arnosti for their generous loan of laboratory equipment. We also thank Barbara Makela and Ella Markley for technical support. We thank Dr Melinda Frame and the Center for Advanced Microscopy at Michigan State University for their assistance with confocal microscopy, and thank MSU Campus Animal Resources for maintenance of our laboratory animals.Publication status: PublishedFunder: Michigan State UniversityBone morphogenic protein (BMP) signaling plays an essential and highly conserved role in embryo axial patterning in animal species. However, in mammalian embryos, which develop inside the mother, early development includes a preimplantation stage, which does not occur in externally developing embryos. During preimplantation, the epiblast is segregated from extra-embryonic lineages that enable implantation and development in utero. Yet, the requirement for BMP signaling is imprecisely defined in mouse early embryos. Here, we show that, in contrast to previous reports, BMP signaling (SMAD1/5/9 phosphorylation) is not detectable until implantation when it is detected in the primitive endoderm - an extra-embryonic lineage. Moreover, preimplantation development appears to be normal following deletion of maternal and zygotic Smad4, an essential effector of canonical BMP signaling. In fact, mice lacking maternal Smad4 are viable. Finally, we uncover a new requirement for zygotic Smad4 in epiblast scaling and cavitation immediately after implantation, via a mechanism involving FGFR/ERK attenuation. Altogether, our results demonstrate no role for BMP4/SMAD4 in the first lineage decisions during mouse development. Rather, multi-pathway signaling among embryonic and extra-embryonic cell types drives epiblast morphogenesis postimplantation

<i>Sox2</i> is required to maintain expression EPI, but not most PE genes.

<p>A) By E4.25, expression of PE genes, including SOX17, PDGFRA, and GATA4, is restored in <i>Sox2</i> null embryos, but the ICM appears disorganized relative to control embryos. (arrowheads  =  mislocalized PE cells. B) In <i>Sox2</i> null embryos, the average proportion of ICM cells expressing SOX17 increases progressively, catching up with control embryos by E4.25. C) Quantification of the average number of apoptotic cells in wild type and <i>Sox2</i> null embryos at the indicated time points. D) At E4.25, the expression of SOX17 in <i>Sox2</i> null embryos depends on FGFR/MEK. E) At E4.25, SOX7, a marker of mature PE, is detectable in the absence of <i>Sox2</i>. F) At E4.25, expression of LAMA1 and DAB2 are reduced in the absence of <i>Sox2</i>, consistent with defects in PE localization. G) At E4.25, expression of OCT4, PECAM1, and NANOG are reduced in the absence of <i>Sox2</i>. Bar  = 20 µm, p-value calculated by t-test; n.s.  = p>0.05.</p

SOX2 is restricted to ICM progenitors by HIPPO pathway members and not by CDX2.

<p>A) Immunofluorescent analysis of SOX2 and NANOG shows that SOX2 is detected specifically in ICM cells at the 16-cell stage and later, while NANOG is detected in all cells at these stages. B) SOX2 is not upregulated in the TE of <i>Cdx2</i> null embryos at early or late blastocyst stages, indicating that CDX2 does not restrict SOX2 to the ICM. C) SOX2 is ectopically expressed in outside cells of embryos lacking the HIPPO pathway member <i>Tead4</i> (asterisk  =  SOX2-positive outside cell). TE cells are defined both by outside position and by basolateral localization of E-CADHERIN (ECAD). D) Either <i>Lats2</i> or <i>β-Globin</i> mRNAs were injected into both cells of 2-cell embryos, and embryos were then cultured to blastocyst stage. E) The proportion of outside cells in which SOX2 was ectopically expressed was significantly increased in both <i>Tead4</i> null embryos, and in embryos overexpressing the HIPPO pathway member <i>Lats2</i>, relative to controls (p-value calculated by t-test). F) Overexpression of <i>Lats2</i>, which prevents nuclear YAP localization, causes ectopic expression of SOX2 in outside cells (indicated by asterisk). In all panels, bar  = 20 µm.</p