Human prefrontal cortex gene regulatory dynamics from gestation to adulthood at single-cell resolution.

Human brain development is underpinned by cellular and molecular reconfigurations continuing into the third decade of life. To reveal cell dynamics orchestrating neural maturation, we profiled human prefrontal cortex gene expression and chromatin accessibility at single-cell resolution from gestation to adulthood. Integrative analyses define the dynamic trajectories of each cell type, revealing major gene expression reconfiguration at the prenatal-to-postnatal transition in all cell types followed by continuous reconfiguration into adulthood and identifying regulatory networks guiding cellular developmental programs, states, and functions. We uncover links between expression dynamics and developmental milestones, characterize the diverse timing of when cells acquire adult-like states, and identify molecular convergence from distinct developmental origins. We further reveal cellular dynamics and their regulators implicated in neurological disorders. Finally, using this reference, we benchmark cell identities and maturation states in organoid models. Together, this captures the dynamic regulatory landscape of human cortical development.

A modular dCas9-based recruitment platform for combinatorial epigenome editing.

Targeted epigenome editing tools allow precise manipulation and investigation of genome modifications, however they often display high context dependency and variable efficacy between target genes and cell types. While systems that simultaneously recruit multiple distinct 'effector' chromatin regulators can improve efficacy, they generally lack control over effector composition and spatial organisation. To overcome this we have created a modular combinatorial epigenome editing platform, called SSSavi. This system is an interchangeable and reconfigurable docking platform fused to dCas9 that enables simultaneous recruitment of up to four different effectors, allowing precise control of effector composition and spatial ordering. We demonstrate the activity and specificity of the SSSavi system and, by testing it against existing multi-effector targeting systems, demonstrate its comparable efficacy. Furthermore, we demonstrate the importance of the spatial ordering of the recruited effectors for effective transcriptional regulation. Together, the SSSavi system enables exploration of combinatorial effector co-recruitment to enhance manipulation of chromatin contexts previously resistant to targeted editing.

A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs.

Detection of DNA methylation in the genome has been possible for decades; however, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded our understanding of the direct effect of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this. Recent adaptations of genome editing technologies, including fusion of the DNMT3A DNA methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to alter DNA methylation at desired loci. Here, we show that these tools exhibit consistent off-target DNA methylation deposition in the genome, limiting their capabilities to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA methylation. dC9Sun-D3A is tunable, specific, and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target methylation by dC9-D3A. Furthermore, we used dC9Sun-D3A to demonstrate the binding sensitivity to DNA methylation for CTCF and NRF1 in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest off-target DNA methylation levels reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.

Age-associated sperm DNA methylation alterations: possible implications in offspring disease susceptibility.

Recent evidence demonstrates a role for paternal aging on offspring disease susceptibility. It is well established that various neuropsychiatric disorders (schizophrenia, autism, etc.), trinucleotide expansion associated diseases (myotonic dystrophy, Huntington's, etc.) and even some forms of cancer have increased incidence in the offspring of older fathers. Despite strong epidemiological evidence that these alterations are more common in offspring sired by older fathers, in most cases the mechanisms that drive these processes are unclear. However, it is commonly believed that epigenetics, and specifically DNA methylation alterations, likely play a role. In this study we have investigated the impact of aging on DNA methylation in mature human sperm. Using a methylation array approach we evaluated changes to sperm DNA methylation patterns in 17 fertile donors by comparing the sperm methylome of 2 samples collected from each individual 9-19 years apart. With this design we have identified 139 regions that are significantly and consistently hypomethylated with age and 8 regions that are significantly hypermethylated with age. A representative subset of these alterations have been confirmed in an independent cohort. A total of 117 genes are associated with these regions of methylation alterations (promoter or gene body). Intriguingly, a portion of the age-related changes in sperm DNA methylation are located at genes previously associated with schizophrenia and bipolar disorder. While our data does not establish a causative relationship, it does raise the possibility that the age-associated methylation of the candidate genes that we observe in sperm might contribute to the increased incidence of neuropsychiatric and other disorders in the offspring of older males. However, further study is required to determine whether, and to what extent, a causative relationship exists.

Interaction of the Jhd2 Histone H3 Lys-4 Demethylase with Chromatin Is Controlled by Histone H2A Surfaces and Restricted by H2B Ubiquitination.

Histone H3 lysine 4 (H3K4) methylation is a dynamic modification. In budding yeast, H3K4 methylation is catalyzed by the Set1-COMPASS methyltransferase complex and is removed by Jhd2, a JMJC domain family demethylase. The catalytic JmjC and JmjN domains of Jhd2 have the ability to remove all three degrees (mono-, di-, and tri-) of H3K4 methylation. Jhd2 also contains a plant homeodomain (PHD) finger required for its chromatin association and H3K4 demethylase functions. The Jhd2 PHD finger associates with chromatin independent of H3K4 methylation and the H3 N-terminal tail. Therefore, how Jhd2 associates with chromatin to perform H3K4 demethylation has remained unknown. We report a novel interaction between the Jhd2 PHD finger and histone H2A. Two residues in H2A (Phe-26 and Glu-57) serve as a binding site for Jhd2 in vitro and mediate its chromatin association and H3K4 demethylase functions in vivo. Using RNA sequencing, we have identified the functional target genes for Jhd2 and the H2A Phe-26 and Glu-57 residues. We demonstrate that H2A Phe-26 and Glu-57 residues control chromatin association and H3K4 demethylase functions of Jhd2 during positive or negative regulation of transcription at target genes. Importantly, we show that H2B Lys-123 ubiquitination blocks Jhd2 from accessing its binding site on chromatin, and thereby, we have uncovered a second mechanism by which H2B ubiquitination contributes to the trans-histone regulation of H3K4 methylation. Overall, our study provides novel insights into the chromatin binding dynamics and H3K4 demethylase functions of Jhd2.

CRISPRi-based circuits to control gene expression in plants.

The construction of synthetic gene circuits in plants has been limited by a lack of orthogonal and modular parts. Here, we implement a CRISPR (clustered regularly interspaced short palindromic repeats) interference (CRISPRi)-based reversible gene circuit platform in plants. We create a toolkit of engineered repressible promoters of different strengths and construct NOT and NOR gates in Arabidopsis thaliana protoplasts. We determine the optimal processing system to express single guide RNAs from RNA Pol II promoters to introduce NOR gate programmability for interfacing with host regulatory sequences. The performance of a NOR gate in stably transformed Arabidopsis plants demonstrates the system's programmability and reversibility in a complex multicellular organism. Furthermore, cross-species activity of CRISPRi-based logic gates is shown in Physcomitrium patens, Triticum aestivum and Brassica napus protoplasts. Layering multiple NOR gates together creates OR, NIMPLY and AND logic functions, highlighting the modularity of our system. Our CRISPRi circuits are orthogonal, compact, reversible, programmable and modular and provide a platform for sophisticated spatiotemporal control of gene expression in plants.

Synthetic Epigenetic Reprogramming of Mesenchymal to Epithelial States Using the CRISPR/dCas9 Platform in Triple Negative Breast Cancer.

Epithelial-mesenchymal transition (EMT) is a reversible transcriptional program invoked by cancer cells to drive cancer progression. Transcription factor ZEB1 is a master regulator of EMT, driving disease recurrence in poor-outcome triple negative breast cancers (TNBCs). Here, this work silences ZEB1 in TNBC models by CRISPR/dCas9-mediated epigenetic editing, resulting in highly-specific and nearly complete suppression of ZEB1 in vivo, accompanied by long-lasting tumor inhibition. Integrated "omic" changes promoted by dCas9 linked to the KRAB domain (dCas9-KRAB) enabled the discovery of a ZEB1-dependent-signature of 26 genes differentially-expressed and -methylated, including the reactivation and enhanced chromatin accessibility in cell adhesion loci, outlining epigenetic reprogramming toward a more epithelial state. In the ZEB1 locus transcriptional silencing is associated with induction of locally-spread heterochromatin, significant changes in DNA methylation at specific CpGs, gain of H3K9me3, and a near complete erasure of H3K4me3 in the ZEB1 promoter. Epigenetic shifts induced by ZEB1-silencing are enriched in a subset of human breast tumors, illuminating a clinically-relevant hybrid-like state. Thus, the synthetic epi-silencing of ZEB1 induces stable "lock-in" epigenetic reprogramming of mesenchymal tumors associated with a distinct and stable epigenetic landscape. This work outlines epigenome-engineering approaches for reversing EMT and customizable precision molecular oncology approaches for targeting poor outcome breast cancers.

Synthetic memory circuits for stable cell reprogramming in plants.

Plant biotechnology predominantly relies on a restricted set of genetic parts with limited capability to customize spatiotemporal and conditional expression patterns. Synthetic gene circuits have the potential to integrate multiple customizable input signals through a processing unit constructed from biological parts to produce a predictable and programmable output. Here we present a suite of functional recombinase-based gene circuits for use in plants. We first established a range of key gene circuit components compatible with plant cell functionality. We then used these to develop a range of operational logic gates using the identify function (activation) and negation function (repression) in Arabidopsis protoplasts and in vivo, demonstrating their utility for programmable manipulation of transcriptional activity in a complex multicellular organism. Specifically, using recombinases and plant control elements, we activated transgenes in YES, OR and AND gates and repressed them in NOT, NOR and NAND gates; we also implemented the A NIMPLY B gate that combines activation and repression. Through use of genetic recombination, these circuits create stable long-term changes in expression and recording of past stimuli. This highly compact programmable gene circuit platform provides new capabilities for engineering sophisticated transcriptional programs and previously unrealized traits into plants.

Harnessing targeted DNA methylation and demethylation using dCas9.

DNA methylation is an essential DNA modification that plays a crucial role in genome regulation during differentiation and development, and is disrupted in a range of disease states. The recent development of CRISPR/catalytically dead CRISPR/Cas9 (dCas9)-based targeted DNA methylation editing tools has enabled new insights into the roles and functional relevance of this modification, including its importance at regulatory regions and the role of aberrant methylation in various diseases. However, while these tools are advancing our ability to understand and manipulate this regulatory layer of the genome, they still possess a variety of limitations in efficacy, implementation, and targeting specificity. Effective targeted DNA methylation editing will continue to advance our fundamental understanding of the role of this modification in different genomic and cellular contexts, and further improvements may enable more accurate disease modeling and possible future treatments. In this review, we discuss strategies, considerations, and future directions for targeted DNA methylation editing.

Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons.

To better understand transcriptional regulation during human oogenesis and preimplantation development, we defined stage-specific transcription, which highlighted the cleavage stage as being highly distinctive. Here, we present multiple lines of evidence that a eutherian-specific multicopy retrogene, DUX4, encodes a transcription factor that activates hundreds of endogenous genes (for example, ZSCAN4, KDM4E and PRAMEF-family genes) and retroviral elements (MERVL/HERVL family) that define the cleavage-specific transcriptional programs in humans and mice. Remarkably, mouse Dux expression is both necessary and sufficient to convert mouse embryonic stem cells (mESCs) into 2-cell-embryo-like ('2C-like') cells, measured here by the reactivation of '2C' genes and repeat elements, the loss of POU5F1 (also known as OCT4) protein and chromocenters, and the conversion of the chromatin landscape (as assessed by transposase-accessible chromatin using sequencing (ATAC-seq)) to a state strongly resembling that of mouse 2C embryos. Thus, we propose mouse DUX and human DUX4 as major drivers of the cleavage or 2C state.

Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription.

Histone H3K4 methylation is connected to gene transcription from yeast to humans, but its mechanistic roles in transcription and chromatin dynamics remain poorly understood. We investigated the functions for Set1 and Jhd2, the sole H3K4 methyltransferase and H3K4 demethylase, respectively, in S. cerevisiae. Here, we show that Set1 and Jhd2 predominantly co-regulate genome-wide transcription. We find combined activities of Set1 and Jhd2 via H3K4 methylation contribute to positive or negative transcriptional regulation. Providing mechanistic insights, our data reveal that Set1 and Jhd2 together control nucleosomal turnover and occupancy during transcriptional co-regulation. Moreover, we find a genome-wide co-regulation of chromatin structure by Set1 and Jhd2 at different groups of transcriptionally active or inactive genes and at different regions within yeast genes. Overall, our study puts forth a model wherein combined actions of Set1 and Jhd2 via modulating H3K4 methylation-demethylation together control chromatin dynamics during various facets of transcriptional regulation.

Alterations in sperm DNA methylation patterns at imprinted loci in two classes of infertility.

OBJECTIVE: To evaluate the associations between proper protamine incorporation and DNA methylation at imprinted loci. DESIGN: Experimental research study. SETTING: Research laboratory. PATIENT(S): Three populations were tested-abnormal protamine patients, oligozoospermic patients, and fertile donors. INTERVENTION(S): The CpG methylation patterns were examined at seven imprinted loci sequenced: LIT1, MEST, SNRPN, PLAGL1, PEG3, H19, and IGF2. MAIN OUTCOME MEASURE(S): The DNA methylation patterns were analyzed using bisulfite sequencing. The percentage of methylation was compared between fertile and infertile patients displaying abnormal protamination. RESULT(S): At six of the seven imprinted genes, the overall DNA methylation patterns at their respective differentially methylated regions were significantly altered in both infertile patient populations. When comparing the severity of methylation alterations among infertile patients, the oligozoospermic patients were significantly affected at mesoderm-specific transcript (MEST), whereas abnormal protamine patients were affected at KCNQ1, overlapping transcript 1 (LIT1), and at small nuclear ribonucleoprotein polypeptide N (SNRPN). CONCLUSION(S): Patients with male factor infertility had significantly increased methylation alteration at six of seven imprinted loci tested, with differences in significance observed between oligozoospermic and abnormal protamine patients. This could suggest that risk of transmission of epigenetic alterations may be different with diagnoses. However, this study does not provide a causal link for epigenetic inheritance of imprinting diseases, but does show significant association between male factor infertility and alterations in sperm DNA methylation at imprinted loci.