Diverse heterochromatin states restricting cell identity and reprogramming.

Heterochromatin is defined as a chromosomal domain harboring repressive H3K9me2/3 or H3K27me3 histone modifications and relevant factors that physically compact the chromatin. Heterochromatin can restrict where transcription factors bind, providing a barrier to gene activation and changes in cell identity. While heterochromatin thus helps maintain cell differentiation, it presents a barrier to overcome during efforts to reprogram cells for biomedical purposes. Recent findings have revealed complexity in the composition and regulation of heterochromatin, and shown that transiently disrupting the machinery of heterochromatin can enhance reprogramming. Here, we discuss how heterochromatin is established and maintained during development, and how our growing understanding of the mechanisms regulating H3K9me3 heterochromatin can be leveraged to improve our ability to direct changes in cell identity.

A pioneer factor locally opens compacted chromatin to enable targeted ATP-dependent nucleosome remodeling.

To determine how different pioneer transcription factors form a targeted, accessible nucleosome within compacted chromatin and collaborate with an ATP-dependent chromatin remodeler, we generated nucleosome arrays in vitro with a central nucleosome containing binding sites for the hematopoietic E-Twenty Six (ETS) factor PU.1 and Basic Leucine Zipper (bZIP) factors C/EBPα and C/EBPß. Our long-read sequencing reveals that each factor can expose a targeted nucleosome on linker histone-compacted arrays, but with different nuclease sensitivity patterns. The DNA binding domain of PU.1 binds mononucleosomes, but requires an additional intrinsically disordered domain to bind and open compacted chromatin. The canonical mammalian SWI/SNF (cBAF) remodeler was unable to act upon two forms of locally open chromatin unless cBAF was enabled by a separate transactivation domain of PU.1. cBAF potentiates the PU.1 DNA binding domain to weakly open chromatin in the absence of the PU.1 disordered domain. Our findings reveal a hierarchy by which chromatin is opened and show that pioneer factors can provide specificity for action by nucleosome remodelers.

Diverse heterochromatin-associated proteins repress distinct classes of genes and repetitive elements.

Heterochromatin, typically marked by histone H3 trimethylation at lysine 9 (H3K9me3) or lysine 27 (H3K27me3), represses different protein-coding genes in different cells, as well as repetitive elements. The basis for locus specificity is unclear. Previously, we identified 172 proteins that are embedded in sonication-resistant heterochromatin (srHC) harbouring H3K9me3. Here, we investigate in humans how 97 of the H3K9me3-srHC proteins repress heterochromatic genes. We reveal four groups of srHC proteins that each repress many common genes and repeat elements. Two groups repress H3K9me3-embedded genes with different extents of flanking srHC, one group is specific for srHC genes with H3K9me3 and H3K27me3, and one group is specific for genes with srHC as the primary feature. We find that the enhancer of rudimentary homologue (ERH) is conserved from Schizosaccharomyces pombe in repressing meiotic genes and, in humans, now represses other lineage-specific genes and repeat elements. The study greatly expands our understanding of H3K9me3-based gene repression in vertebrates.

Two-parameter single-molecule analysis for measurement of chromatin mobility.

This protocol provides a two-parameter analysis of single-molecule tracking (SMT) trajectories of Halo-tagged histones in living adherent cell lines and unveils a chromatin mobility landscape composed of five chromatin types, ranging from low to high mobility. When the analysis is applied to Halo-tagged, chromatin-binding proteins, it associates chromatin interaction properties with known functions in a way that previously used SMT parameters did not. For complete information on the use and execution of this protocol, please refer to Lerner et al. (2020).

Two-Parameter Mobility Assessments Discriminate Diverse Regulatory Factor Behaviors in Chromatin.

Enzymatic probes of chromatin structure reveal accessible versus inaccessible chromatin states, while super-resolution microscopy reveals a continuum of chromatin compaction states. Characterizing histone H2B movements by single-molecule tracking (SMT), we resolved chromatin domains ranging from low to high mobility and displaying different subnuclear localizations patterns. Heterochromatin constituents correlated with the lowest mobility chromatin, whereas transcription factors varied widely with regard to their respective mobility with low- or high-mobility chromatin. Pioneer transcription factors, which bind nucleosomes, can access the low-mobility chromatin domains, whereas weak or non-nucleosome binding factors are excluded from the domains and enriched in higher mobility domains. Nonspecific DNA and nucleosome binding accounted for most of the low mobility of strong nucleosome interactor FOXA1. Our analysis shows how the parameters of the mobility of chromatin-bound factors, but not their diffusion behaviors or SMT-residence times within chromatin, distinguish functional characteristics of different chromatin-interacting proteins.

Genomic and Proteomic Resolution of Heterochromatin and Its Restriction of Alternate Fate Genes.

Heterochromatin is integral to cell identity maintenance by impeding the activation of genes for alternate cell fates. Heterochromatic regions are associated with histone 3 lysine 9 trimethylation (H3K9me3) or H3K27me3, but these modifications are also found in euchromatic regions that permit transcription. We discovered that resistance to sonication is a reliable indicator of the heterochromatin state, and we developed a biophysical method (gradient-seq) to discriminate subtypes of H3K9me3 and H3K27me3 domains in sonication-resistant heterochromatin (srHC) versus euchromatin. These classifications are more accurate than the histone marks alone in predicting transcriptional silence and resistance of alternate fate genes to activation during direct cell conversion. Our proteomics of H3K9me3-marked srHC and functional screens revealed diverse proteins, including RBMX and RBMXL1, that impede gene induction during cellular reprogramming. Isolation of srHC with gradient-seq provides a genome-wide map of chromatin structure, elucidating subtypes of repressed domains that are uniquely predictive of diverse other chromatin properties.

Rapid monoisotopic cisplatin based barcoding for multiplexed mass cytometry.

Mass cytometry presents an exceptional opportunity to interrogate the biology of highly heterogeneous cell populations, owing to the ability to collect highly parametric proteomic data at a single cell level. However, sample-to-sample variability, due to antibody staining and/or instrument sensitivity, can introduce substantial artifacts into the data, which can in turn lead to erroneous conclusions. This variability can be eliminated by sample barcoding which enables samples to be pooled, stained and run simultaneously. Existing mass cytometry barcoding approaches require time intensive labeling, reduce the number of biologically meaningful parameters and/or rely on expensive reagents. We present an approach utilizing monoisotopic cisplatin to perform cell barcoding that does not require cell permeabilization, can be completed in 10 minutes and can be utilized in combination with existing barcoding techniques to greatly increase the number of samples which can be multiplexed to improve throughput and consistency.

Sample Preparation for Mass Cytometry Analysis.

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

MicroRNA Regulates Hepatocytic Differentiation of Progenitor Cells by Targeting YAP1.

MicroRNA expression profiling in human liver progenitor cells following hepatocytic differentiation identified miR-122 and miR-194 as the microRNAs most strongly upregulated during hepatocytic differentiation of progenitor cells. MiR-194 was also highly upregulated following hepatocytic differentiation of human embryonic stem cells (hESCs). Overexpression of miR-194 in progenitor cells accelerated their differentiation into hepatocytes, as measured by morphological features such as canaliculi and expression of hepatocytic markers. Overexpression of miR-194 in hESCs induced their spontaneous differentiation, a phenotype accompanied with accelerated loss of the pluripotent factors OCT4 and NANOG and decrease in mesoderm marker HAND1 expression. We then identified YAP1 as a direct target of miR-194. Inhibition of YAP1 strongly induced hepatocytic differentiation of progenitor cells and YAP1 overexpression reversed the miR-194-induced hepatocytic differentiation of progenitor cells. In conclusion, we identified miR-194 as a potent inducer of hepatocytic differentiation of progenitor cells and further identified YAP1 as a mediator of miR-194's effects on hepatocytic differentiation and liver progenitor cell fate. Stem Cells 2016;34:1284-1296.

The poplar MYB master switches bind to the SMRE site and activate the secondary wall biosynthetic program during wood formation.

Wood is mainly composed of secondary walls, which constitute the most abundant stored carbon produced by vascular plants. Understanding the molecular mechanisms controlling secondary wall deposition during wood formation is not only an important issue in plant biology but also critical for providing molecular tools to custom-design wood composition suited for diverse end uses. Past molecular and genetic studies have revealed a transcriptional network encompassing a group of wood-associated NAC and MYB transcription factors that are involved in the regulation of the secondary wall biosynthetic program during wood formation in poplar trees. Here, we report the functional characterization of poplar orthologs of MYB46 and MYB83 that are known to be master switches of secondary wall biosynthesis in Arabidopsis. In addition to the two previously-described PtrMYB3 and PtrMYB20, two other MYBs, PtrMYB2 and PtrMYB21, were shown to be MYB46/MYB83 orthologs by complementation and overexpression studies in Arabidopsis. The functional roles of these PtrMYBs in regulating secondary wall biosynthesis were further demonstrated in transgenic poplar plants showing an ectopic deposition of secondary walls in PtrMYB overexpressors and a reduction of secondary wall thickening in their dominant repressors. Furthermore, PtrMYB2/3/20/21 together with two other tree MYBs, the Eucalyptus EgMYB2 and the pine PtMYB4, were shown to differentially bind to and activate the eight variants of the 7-bp SMRE consensus sequence, composed of ACC(A/T)A(A/C)(T/C). Together, our results indicate that the tree MYBs, PtrMYB2/3/20/21, EgMYB2 and PtMYB4, are master transcriptional switches that activate the SMRE sites in the promoters of target genes and thereby regulate secondary wall biosynthesis during wood formation.

Transcriptional activation of secondary wall biosynthesis by rice and maize NAC and MYB transcription factors.

The bulk of grass biomass potentially useful for cellulose-based biofuel production is the remains of secondary wall-containing sclerenchymatous fibers. Hence, it is important to uncover the molecular mechanisms underlying the regulation of secondary wall thickening in grass species. So far, little is known about the transcriptional regulatory switches responsible for the activation of the secondary wall biosynthetic program in grass species. Here, we report the roles of a group of rice and maize NAC and MYB transcription factors in the regulation of secondary wall biosynthesis. The rice and maize secondary wall-associated NACs (namely OsSWNs and ZmSWNs) were able to complement the Arabidopsis snd1 nst1 double mutant defective in secondary wall thickening. When overexpressed in Arabidopsis, OsSWNs and ZmSWNs were sufficient to activate a number of secondary wall-associated transcription factors and secondary wall biosynthetic genes, and concomitantly result in the ectopic deposition of cellulose, xylan and lignin. It was also found that the rice and maize MYB transcription factors, OsMYB46 and ZmMYB46, are functional orthologs of Arabidopsis MYB46/MYB83 and, when overexpressed in Arabidopsis, they were able to activate the entire secondary wall biosynthetic program. Furthermore, the promoters of OsMYB46 and ZmMYB46 contain secondary wall NAC-binding elements (SNBEs), which can be bound and activated by OsSWNs and ZmSWNs. Together, our results indicate that the rice and maize SWNs and MYB46 are master transcriptional activators of the secondary wall biosynthetic program and that OsSWNs and ZmSWNs activate their direct target genes through binding to the SNBE sites.

Dissection of the transcriptional program regulating secondary wall biosynthesis during wood formation in poplar.

Wood biomass is mainly made of secondary cell walls; hence, elucidation of the molecular mechanisms underlying the transcriptional regulation of secondary wall biosynthesis during wood formation will be instrumental to design strategies for genetic improvement of wood biomass. Here, we provide direct evidence demonstrating that the poplar (Populus trichocarpa) wood-associated NAC domain transcription factors (PtrWNDs) are master switches activating a suite of downstream transcription factors, and together, they are involved in the coordinated regulation of secondary wall biosynthesis during wood formation. We show that transgenic poplar plants with dominant repression of PtrWNDs functions exhibit a drastic reduction in secondary wall thickening in woody cells, and those with PtrWND overexpression result in ectopic deposition of secondary walls. Analysis of PtrWND2B overexpressors revealed up-regulation of the expression of a number of wood-associated transcription factors, the promoters of which were also activated by PtrWND6B and the Eucalyptus EgWND1. Transactivation analysis and electrophoretic mobility shift assay demonstrated that PtrWNDs and EgWND1 activated gene expression through direct binding to the secondary wall NAC-binding elements, which are present in the promoters of several wood-associated transcription factors and a number of genes involved in secondary wall biosynthesis and modification. The WND-regulated transcription factors PtrNAC150, PtrNAC156, PtrNAC157, PtrMYB18, PtrMYB74, PtrMYB75, PtrMYB121, PtrMYB128, PtrZF1, and PtrGATA8 were able to activate the promoter activities of the biosynthetic genes for all three major wood components. Our study has uncovered that the WND master switches together with a battery of their downstream transcription factors form a transcriptional network controlling secondary wall biosynthesis during wood formation.

Secondary wall NAC binding element (SNBE), a key cis-acting element required for target gene activation by secondary wall NAC master switches.

The biosynthesis of secondary walls in vascular plants requires the coordinated regulation of a suite of biosynthetic genes, and this coordination has recently been shown to be executed by the secondary wall NAC (SWN)-mediated transcriptional network. In Arabidopsis, five SWNs, including SND1, NST1/2 and VND6/7, function as master transcriptional switches to activate their common targets and consequently the secondary wall biosynthetic program. A recent report by Zhong et al. revealed that SWNs bind to a common cis-acting element, namely secondary wall NAC binding element (SNBE), which is composed of an imperfect palindromic 19-bp consensus sequence, (T/A)NN(C/T)(T/C/G)TNNNNNNNA(A/C)GN(A/C/T) (A/T). Genome-wide analysis of direct targets of SWNs showed that SWNs directly activate the expression of not only many transcription factors but also a battery of genes involved in secondary wall biosynthesis, cell wall modification and programmed cell death, the promoters of which all contain multiple SNBE sites. The functional significance of the SNBE sites is further substantiated by our current in planta expression study demonstrating that representative SNBE sequences from several SWN direct target promoters are sufficient to drive the expression of the GUS reporter gene in secondary wall-forming cells. The identification of the SWN DNA binding element (SNBE) and the SWN direct targets marks an important step forward toward the dissection of the transcriptional network regulating the biosynthesis of secondary walls, the most abundant biomass produced by land plants.

The poplar MYB transcription factors, PtrMYB3 and PtrMYB20, are involved in the regulation of secondary wall biosynthesis.

Dicot wood is mainly composed of cellulose, xylan and lignin, and its formation requires the coordinated regulation of their biosynthesis. In this report, we demonstrate that the poplar wood-associated MYB transcriptional activators, PtrMYB3 and PtrMYB20, activate the biosynthetic pathways of cellulose, xylan and lignin when overexpressed in Arabidopsis and they are also able to activate the promoter activities of poplar wood biosynthetic genes. We also show that PtrMYB3 and PtrMYB20 are functional orthologs of Arabidopsis MYB46 and MYB83, and their expression is directly activated by poplar PtrWND2, suggesting their involvement in the regulation of wood formation in poplar.

MYB83 is a direct target of SND1 and acts redundantly with MYB46 in the regulation of secondary cell wall biosynthesis in Arabidopsis.

It has been proposed that the transcriptional regulation of secondary wall biosynthesis in Arabidopsis is controlled by a transcriptional network mediated by SND1 and its close homologs. Uncovering all the transcription factors and deciphering their interrelationships in the network are essential for our understanding of the molecular mechanisms underlying the transcriptional regulation of biosynthesis of secondary walls, the major constituent of wood and fibers. Here, we present functional evidence that the MYB83 transcription factor is another molecular switch in the SND1-mediated transcriptional network regulating secondary wall biosynthesis. MYB83 is specifically expressed in fibers and vessels where secondary wall thickening occurs. Its expression is directly activated by SND1 and its close homologs, including NST1, NST2, VND6 and VND7, indicating that MYB83 is their direct target. MYB83 overexpression is able to activate a number of the biosynthetic genes of cellulose, xylan and lignin and concomitantly induce ectopic secondary wall deposition. In addition, its overexpression upregulates the expression of several transcription factors involved in regulation of secondary wall biosynthesis. Dominant repression of MYB83 functions or simultaneous RNAi inhibition of MYB83 and MYB46 results in a reduction in secondary wall thickening in fibers and vessels and a deformation of vessels. Furthermore, double T-DNA knockout mutations of MYB83 and MYB46 cause a lack of secondary walls in vessels and an arrest in plant growth. Together, these results demonstrate that MYB83 and MYB46, both of which are SND1 direct targets, function redundantly in the transcriptional regulatory cascade leading to secondary wall formation in fibers and vessels.

A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis.

SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1) is a master transcriptional switch activating the developmental program of secondary wall biosynthesis. Here, we demonstrate that a battery of SND1-regulated transcription factors is required for normal secondary wall biosynthesis in Arabidopsis thaliana. The expression of 11 SND1-regulated transcription factors, namely, SND2, SND3, MYB103, MYB85, MYB52, MYB54, MYB69, MYB42, MYB43, MYB20, and KNAT7 (a Knotted1-like homeodomain protein), was developmentally associated with cells undergoing secondary wall thickening. Of these, dominant repression of SND2, SND3, MYB103, MYB85, MYB52, MYB54, and KNAT7 significantly reduced secondary wall thickening in fiber cells. Overexpression of SND2, SND3, and MYB103 increased secondary wall thickening in fibers, and overexpression of MYB85 led to ectopic deposition of lignin in epidermal and cortical cells in stems. Furthermore, SND2, SND3, MYB103, MYB85, MYB52, and MYB54 were able to induce secondary wall biosynthetic genes. Direct target analysis using the estrogen-inducible system revealed that MYB46, SND3, MYB103, and KNAT7 were direct targets of SND1 and also of its close homologs, NST1, NST2, and vessel-specific VND6 and VND7. Together, these results demonstrate that a transcriptional network consisting of SND1 and its downstream targets is involved in regulating secondary wall biosynthesis in fibers and that NST1, NST2, VND6, and VND7 are functional homologs of SND1 that regulate the same downstream targets in different cell types.