Dual-role transcription factors stabilize intermediate expression levels.

Precise control of gene expression levels is essential for normal cell functions, yet how they are defined and tightly maintained, particularly at intermediate levels, remains elusive. Here, using a series of newly developed sequencing, imaging, and functional assays, we uncover a class of transcription factors with dual roles as activators and repressors, referred to as condensate-forming level-regulating dual-action transcription factors (TFs). They reduce high expression but increase low expression to achieve stable intermediate levels. Dual-action TFs directly exert activating and repressing functions via condensate-forming domains that compartmentalize core transcriptional unit selectively. Clinically relevant mutations in these domains, which are linked to a range of developmental disorders, impair condensate selectivity and dual-action TF activity. These results collectively address a fundamental question in expression regulation and demonstrate the potential of level-regulating dual-action TFs as powerful effectors for engineering controlled expression levels.

G9a/GLP-sensitivity of H3K9me2 Demarcates Two Types of Genomic Compartments.

In the nucleus, chromatin is folded into hierarchical architecture that is tightly linked to various nuclear functions. However, the underlying molecular mechanisms that confer these architectures remain incompletely understood. Here, we investigated the functional roles of H3 lysine 9 dimethylation (H3K9me2), one of the abundant histone modifications, in three-dimensional (3D) genome organization. Unlike in mouse embryonic stem cells, inhibition of methyltransferases G9a and GLP in differentiated cells eliminated H3K9me2 predominantly at A-type (active) genomic compartments, and the level of residual H3K9me2 modifications was strongly associated with B-type (inactive) genomic compartments. Furthermore, chemical inhibition of G9a/GLP in mouse hepatocytes led to decreased chromatin-nuclear lamina interactions mainly at G9a/GLP-sensitive regions, increased degree of genomic compartmentalization, and up-regulation of hundreds of genes that were associated with alterations of the 3D chromatin. Collectively, our data demonstrated essential roles of H3K9me2 in 3D genome organization.

TOPORS, a tumor suppressor protein, contributes to the maintenance of higher-order chromatin architecture.

In the nucleus, chromosomes are hierarchically folded into active (A) and inactive (B) compartments composed of topologically associating domains (TADs). Genomic regions interact with nuclear lamina, termed lamina-associated domains (LADs). However, the molecular mechanisms underlying these 3D chromatin architectures remain incompletely understood. Here, we investigated the role of a potential tumor suppressor, TOP1 Binding Arginine/Serine Rich Protein (TOPORS), in genome organization. In mouse hepatocytes, chromatin interactions between A and B compartments increase and compartmentalization strength is reduced significantly upon Topors knockdown. Correspondingly, strength of TAD boundaries located at A/B borders is weakened. In the absence of TOPORS, chromatin-lamina interactions decrease and the coverage of LADs reduces from 53.31% to 46.52%. Interestingly, these changes in 3D genome are associated with PML nuclear bodies and PML-associated domains (PADs). Moreover, chromatin accessibility is altered predominantly at intergenic regions upon Topors knockdown, including a subset of enhancers. These alterations of chromatin are concordant with transcriptome changes, which are associated with carcinogenesis. Collectively, our findings demonstrate that TOPORS functions as a regulator in chromatin structure, providing novel insight into the architectural roles of tumor suppressors in higher-order genome organization.

The Nuclear Matrix Protein SAFB Cooperates with Major Satellite RNAs to Stabilize Heterochromatin Architecture Partially through Phase Separation.

Interphase chromatin is hierarchically organized into higher-order architectures that are essential for gene functions, yet the biomolecules that regulate these 3D architectures remain poorly understood. Here, we show that scaffold attachment factor B (SAFB), a nuclear matrix (NM)-associated protein with RNA-binding functions, modulates chromatin condensation and stabilizes heterochromatin foci in mouse cells. SAFB interacts via its R/G-rich region with heterochromatin-associated repeat transcripts such as major satellite RNAs, which promote the phase separation driven by SAFB. Depletion of SAFB leads to changes in 3D genome organization, including an increase in interchromosomal interactions adjacent to pericentromeric heterochromatin and a decrease in genomic compartmentalization, which could result from the decondensation of pericentromeric heterochromatin. Collectively, we reveal the integrated roles of NM-associated proteins and repeat RNAs in the 3D organization of heterochromatin, which may shed light on the molecular mechanisms of nuclear architecture organization.

The nuclear matrix protein HNRNPU maintains 3D genome architecture globally in mouse hepatocytes.

Eukaryotic chromosomes are folded into higher-order conformations to coordinate genome functions. In addition to long-range chromatin loops, recent chromosome conformation capture (3C)-based studies have indicated higher levels of chromatin structures including compartments and topologically associating domains (TADs), which may serve as units of genome organization and functions. However, the molecular machinery underlying these hierarchically three-dimensional (3D) chromatin architectures remains poorly understood. Via high-throughput assays, including in situ Hi-C, DamID, ChIP-seq, and RNA-seq, we investigated roles of the Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU), a nuclear matrix (NM)-associated protein, in 3D genome organization. Upon the depletion of HNRNPU in mouse hepatocytes, the coverage of lamina-associated domains (LADs) in the genome increases from 53.1% to 68.6%, and a global condensation of chromatin was observed. Furthermore, disruption of HNRNPU leads to compartment switching on 7.5% of the genome, decreases TAD boundary strengths at borders between A (active) and B (inactive) compartments, and reduces chromatin loop intensities. Long-range chromatin interactions between and within compartments or TADs are also significantly remodeled upon HNRNPU depletion. Intriguingly, HNRNPU mainly associates with active chromatin, and 80% of HNRNPU peaks coincide with the binding of CTCF or RAD21. Collectively, we demonstrated that HNRNPU functions as a major factor maintaining 3D chromatin architecture, suggesting important roles of NM-associated proteins in genome organization.