Functional Characterization of the Binding Properties of the Hematopoietic Transcription Factor PU.1

Transcription factors are defined through their ability to recognize and bind specific sequence motifs in genomic DNA. Such sequence motifs are very small compared to the length of the human genome, which is why there is a large proportion of non-functional motifs, where binding of a transcription factor is undesired. The myeloid and B cell-specific transcription factor PU.1 provides a well-suited model to study global dynamic binding processes. This transcription factor is a central regulator of hematopoietic cell differentiation and plays diverse roles in different hematopoietic lineages by regulating cell-type specific genes. How this master transcription factor gains access to its binding sites in the context of chromatin is only partially understood yet. Here, I analyzed its motif cooperativeness and epigenetic regulation and used a transient mRNA-transfection model to study the de novo binding of PU.1 in the lymphatic leukemia cell line CTV-1 that neither expresses PU.1 nor its related ETS-factors SPIB and SPIC. Introduction of PU.1 rapidly initiated a gene expression program (as measured by RNA-sequencing) dominated by myeloid genes which were correlated with PU.1 expression across hematopoietic lineages. ATAC-sequencing revealed extensive remodeling of the chromatin upon PU.1 expression, which was partially associated with the deposition of the histone modification H3K27ac and the enhanced expression of neighboring genes. De novo remodeled sites were significantly associated with clusters of PU.1 sites and/or higher motif scores, suggesting that homotypic binding sites and high affinity consensus sequences are responsible for a large fraction of de novo remodeled PU.1 binding sites. Moreover, shared sites between PU.1 and its ETS-family members ETS-1 and FLI-1 seemed to enhance PU.1’s chromatin remodeling capacity in the lymphoid cell line, likely by establishing novel ETS-dependent co-associations in less wide-open chromatin regions. PU.1 binding in pre-existing open chromatin, however, was predominantly found at single PU.1 binding sites with lower motif scores and many surrounding consensus motifs for other transcription factor families, including GATA and RUNX, which likely enable PU.1 binding at low affinity sites. Titration of PU.1 levels and the analysis of several deletion mutants showed that the efficient binding of PU.1 to de novo remodeled sites was dependent on PU.1 concentration, reduced in the absence of the glutamine-rich domain and even more diminished in the absence of the acidic domain, suggesting that the latter is required for accessing binding sites in closed chromatin. In vivo proximity-dependent biotinylation analysis (BioID) uncovered the association of PU.1 with several components of the SWI/SNF family of chromatin remodeling complexes, including ARID1A, SMARCD2 and SMARCA4 (BRG1) among others. These interactions were specifically lost in the PU.1 mutant lacking the acidic transactivation domain. In conclusion, we could show that the de novo binding of PU.1 to nuclear DNA induces rapid and marked changes in the chromatin landscape of the lymphatic CTV-1 cell line, which requires the acidic transactivation domain and its interaction with the SWI/SNF remodeling complex

The epigenetic pioneer EGR2 initiates DNA demethylation in differentiating monocytes at both stable and transient binding sites

The differentiation of human blood monocytes (MO), the post-mitotic precursors of macrophages (MAC) and dendritic cells (moDC), is accompanied by the active turnover of DNA methylation, but the extent, consequences and mechanisms of DNA methylation changes remain unclear. Here, we profile and compare epigenetic landscapes during IL-4/GM-CSF-driven MO differentiation across the genome and detect several thousand regions that are actively demethylated during culture, both with or without accompanying changes in chromatin accessibility or transcription factor (TF) binding. We further identify TF that are globally associated with DNA demethylation processes. While interferon regulatory factor 4 (IRF4) is found to control hallmark dendritic cell functions with less impact on DNA methylation, early growth response 2 (EGR2) proves essential for MO differentiation as well as DNA methylation turnover at its binding sites. We also show that ERG2 interacts with the 5mC hydroxylase TET2, and its consensus binding sequences show a characteristic DNA methylation footprint at demethylated sites with or without detectable protein binding. Our findings reveal an essential role for EGR2 as epigenetic pioneer in human MO and suggest that active DNA demethylation can be initiated by the TET2-recruiting TF both at stable and transient binding sites.info:eu-repo/semantics/publishedVersio

Mechanisms governing the pioneering and redistribution capabilities of the non-classical pioneer PU.1

Establishing gene regulatory networks during differentiation or reprogramming requires master or pioneer transcription factors (TFs) such as PU.1, a prototype master TF of hematopoietic lineage differentiation. To systematically determine molecular features that control its activity, here we analyze DNA-binding in vitro and genome-wide in vivo across different cell types with native or ectopic PU.1 expression. Although PU.1, in contrast to classical pioneer factors, is unable to access nucleosomal target sites in vitro, ectopic induction of PU.1 leads to the extensive remodeling of chromatin and redistribution of partner TFs. De novo chromatin access, stable binding, and redistribution of partner TFs both require PU.1's N-terminal acidic activation domain and its ability to recruit SWI/SNF remodeling complexes, suggesting that the latter may collect and distribute co-associated TFs in conjunction with the non-classical pioneer TF PU.1

Postmitotic differentiation of human monocytes requires cohesin-structured chromatin

Cohesin is a major structural component of mammalian genomes and is required to maintain loop structures. While acute depletion in short-term culture models suggests a limited importance of cohesin for steady-state transcriptional circuits, long-term studies are hampered by essential functions of cohesin during replication. Here, we study genome architecture in a postmitotic differentiation setting, the differentiation of human blood monocytes (MO). We profile and compare epigenetic, transcriptome and 3D conformation landscapes during MO differentiation (either into dendritic cells or macrophages) across the genome and detect numerous architectural changes, ranging from higher level compartments down to chromatin loops. Changes in loop structures correlate with cohesin-binding, as well as epigenetic and transcriptional changes during differentiation. Functional studies show that the siRNA-mediated depletion of cohesin (and to a lesser extent also CTCF) markedly disturbs loop structures and dysregulates genes and enhancers that are primarily regulated during normal MO differentiation. In addition, gene activation programs in cohesin-depleted MO-derived macrophages are disturbed. Our findings implicate an essential function of cohesin in controlling long-term, differentiation- and activation-associated gene expression programs

Mechanisms of in vivo binding site selection of the hematopoietic master transcription factor PU.1

The transcription factor PU.1 is crucial for the development of many hematopoietic lineages and its binding patterns significantly change during differentiation processes. However, the 'rules' for binding or not-binding of potential binding sites are only partially understood. To unveil basic characteristics of PU.1 binding site selection in different cell types, we studied the binding properties of PU.1 during human macrophage differentiation. Using in vivo and in vitro binding assays, as well as computational prediction, we show that PU.1 selects its binding sites primarily based on sequence affinity, which results in the frequent autonomous binding of high affinity sites in DNase I inaccessible regions (25-45% of all occupied sites). Increasing PU.1 concentrations and the availability of cooperative transcription factor interactions during lineage differentiation both decrease affinity thresholds for in vivo binding and fine-tune cell type-specific PU.1 binding, which seems to be largely independent of DNA methylation. Occupied sites were predominantly detected in active chromatin domains, which are characterized by higher densities of PU.1 recognition sites and neighboring motifs for cooperative transcription factors. Our study supports a model of PU.1 binding control that involves motif-binding affinity, PU.1 concentration, cooperativeness with neighboring transcription factor sites and chromatin domain accessibility, which likely applies to all PU.1 expressing cells

Temporal autoregulation during human PU.1 locus SubTAD formation

Epigenetic control of gene expression occurs within discrete spatial chromosomal units called topologically associating domains (TADs), but the exact spatial requirements of most genes are unknown; this is of particular interest for genes involved in cancer. We therefore applied high-resolution chromosomal conformation capture sequencing to map the three-dimensional (3D) organization of the human locus encoding the key myeloid transcription factor PU.1 in healthy monocytes and acute myeloid leukemia (AML) cells. We identified a dynamic similar to 75-kb unit (SubTAD) as the genomic region in which spatial interactions between PU.1 gene regulatory elements occur during myeloid differentiation and are interrupted in AML. Within this SubTAD, proper initiation of the spatial chromosomal interactions requires PU.1 autoregulation and recruitment of the chromatin-adaptor protein LDB1 (LIM domain-binding protein 1). However, once these spatial interactions have occurred, LDB1 stabilizes them independently of PU.1 autoregulation. Thus, our data support that PU.1 autoregulates its expression in a "hit-and-run" manner by initiating stable chromosomal loops that result in a transcriptionally active chromatin architecture