Nuclear pore complexes (NPCs) are composed of several copies of ~30 different proteins called nucleoporins (Nups). NPCs penetrate the nuclear envelope (NE) and regulate the nucleocytoplasmic trafficking of macromolecules. Beyond this vital role, NPC components influence genome functions in a transport-independent manner. Nups play an evolutionarily conserved role in gene expression regulation that, in metazoans, extends into the nuclear interior. Additionally, in proliferative cells, Nups play a crucial role in genome integrity maintenance and mitotic progression. Here we discuss genome-related functions of Nups and their impact on essential DNA metabolism processes such as transcription, chromosome duplication, and segregation.
Here we show that the Ino80 chromatin remodeling complex (Ino80C) directly prevents euchromatin from invading transcriptionally silent chromatin within intergenic regions and at the border of euchromatin and heterochromatin. Deletion of Ino80C subunits leads to increased H3K79 methylation and noncoding RNA polymerase II (Pol II) transcription centered at the Ino80C-binding sites. The effect of Ino80C is direct, as it blocks H3K79 methylation by Dot1 in vitro. Heterochromatin stimulates the binding of Ino80C in vitro and in vivo. Our data reveal that Ino80C serves as a general silencing complex that restricts transcription to gene units in euchromatin.
To acquire the ability to recognize and destroy virus and plasmid invaders, prokaryotic CRISPR–Cas systems capture fragments of DNA within the host CRISPR locus. Our results indicate that the process of adaptation by a Type II-A CRISPR–Cas system in Streptococcus thermophilus requires Cas1, Cas2, and Csn2. Surprisingly, we found that Cas9, previously identified as the nuclease responsible for ultimate invader destruction, is also essential for adaptation. Cas9 nuclease activity is dispensable for adaptation. In addition, our studies revealed extensive, unbiased acquisition of the self-targeting host genome sequence by the CRISPR–Cas system that is masked in the presence of active target destruction.
Approximately 75% of the human genome is transcribed, the majority of which does not encode protein. However, many noncoding RNAs (ncRNAs) are rapidly degraded after transcription, and relatively few have established functions, questioning the significance of this observation. Here we show that esBAF, a SWI/SNF family nucleosome remodeling factor, suppresses transcription of ncRNAs from ~57,000 nucleosome-depleted regions (NDRs) throughout the genome of mouse embryonic stem cells (ESCs). We show that esBAF functions to both keep NDRs nucleosome-free and promote elevated nucleosome occupancy adjacent to NDRs. Reduction of adjacent nucleosome occupancy upon esBAF depletion is strongly correlated with ncRNA expression, suggesting that flanking nucleosomes form a barrier to pervasive transcription. Upon forcing nucleosome occupancy near two NDRs using a nucleosome-positioning sequence, we found that esBAF is no longer required to silence transcription. Therefore, esBAF’s function to enforce nucleosome occupancy adjacent to NDRs, and not its function to maintain NDRs in a nucleosome-free state, is necessary for silencing transcription over ncDNA. Finally, we show that the ability of a strongly positioned nucleosome to repress ncRNA depends on its translational positioning. These data reveal a novel role for esBAF in suppressing pervasive transcription from open chromatin regions in ESCs.
GLP and G9a are major H3K9 dimethylases and are essential for mouse early embryonic development. GLP and G9a both harbor ankyrin repeat domains that are capable of binding H3K9 methylation. However, the functional significance of their recognition of H3K9 methylation is unknown. Here, we report that the histone methyltransferase activities of GLP and G9a are stimulated by neighboring nucleosomes that are premethylated at H3K9. These stimulation events function in cis and are dependent on the H3K9 methylation binding activities of ankyrin repeat domains of GLP and G9a. Disruption of the H3K9 methylation-binding activity of GLP in mice causes growth retardation of embryos, ossification defects of calvaria, and postnatal lethality due to starvation of the pups. In mouse embryonic stem cells (ESCs) harboring a mutant GLP that lacks H3K9me1-binding activity, critical pluripotent genes, including Oct4 and Nanog, display inefficient establishment of H3K9me2 and delayed gene silencing during differentiation. Collectively, our study reveals a new activation mechanism for GLP and G9a that plays an important role in ESC differentiation and mouse viability.
The transcription factor (TF) interferon regulatory factor 8 (IRF8) controls both developmental and inflammatory stimulus-inducible genes in macrophages, but the mechanisms underlying these two different functions are largely unknown. One possibility is that these different roles are linked to the ability of IRF8 to bind alternative DNA sequences. We found that IRF8 is recruited to distinct sets of DNA consensus sequences before and after lipopolysaccharide (LPS) stimulation. In resting cells, IRF8 was mainly bound to composite sites together with the master regulator of myeloid development PU.1. Basal IRF8–PU.1 binding maintained the expression of a broad panel of genes essential for macrophage functions (such as microbial recognition and response to purines) and contributed to basal expression of many LPS-inducible genes. After LPS stimulation, increased expression of IRF8, other IRFs, and AP-1 family TFs enabled IRF8 binding to thousands of additional regions containing low-affinity multimerized IRF sites and composite IRF–AP-1 sites, which were not premarked by PU.1 and did not contribute to the basal IRF8 cistrome. While constitutively expressed IRF8-dependent genes contained only sites mediating basal IRF8/PU.1 recruitment, inducible IRF8-dependent genes contained variable combinations of constitutive and inducible sites. Overall, these data show at the genome scale how the same TF can be linked to constitutive and inducible gene regulation via distinct combinations of alternative DNA-binding sites.
It is now well established that the E and Id protein axis regulates multiple steps in lymphocyte development. However, it remains unknown how E and Id proteins mechanistically enforce and maintain the naïve T-cell fate. Here we show that Id2 and Id3 suppressed the development and expansion of innate variant follicular helper T (TFH) cells. Innate variant TFH cells required major histocompatibility complex (MHC) class I-like signaling and were associated with germinal center B cells. We found that Id2 and Id3 induced Foxo1 and Foxp1 expression to antagonize the activation of a TFH transcription signature. We show that Id2 and Id3 acted upstream of the Hif1a/Foxo/AKT/mTORC1 pathway as well as the c-myc/p19Arf module to control cellular expansion. We found that mice depleted for Id2 and Id3 expression developed colitis and αβ T-cell lymphomas. Lymphomas depleted for Id2 and Id3 expression displayed elevated levels of c-myc, whereas p19Arf abundance declined. Transcription signatures of Id2- and Id3-depleted lymphomas revealed similarities to genetic deficiencies associated with Burkitt lymphoma. We propose that, in response to antigen receptor and/or cytokine signaling, the E–Id protein axis modulates the activities of the PI3K–AKT–mTORC1–Hif1a and c-myc/p19Arf pathways to control cellular expansion and homeostatic proliferation.
The initiation of chromosome morphogenesis marks the beginning of mitosis in all eukaryotic cells. Although many effectors of chromatin compaction have been reported, the nature and design of the essential trigger for global chromosome assembly remain unknown. Here we reveal the identity of the core mechanism responsible for chromosome morphogenesis in early mitosis. We show that the unique sensitivity of the chromosome condensation machinery for the kinase activity of Cdk1 acts as a major driving force for the compaction of chromatin at mitotic entry. This sensitivity is imparted by multisite phosphorylation of a conserved chromatin-binding sensor, the Smc4 protein. The multisite phosphorylation of this sensor integrates the activation state of Cdk1 with the dynamic binding of the condensation machinery to chromatin. Abrogation of this event leads to chromosome segregation defects and lethality, while moderate reduction reveals the existence of a novel chromatin transition state specific to mitosis, the intertwist configuration. Collectively, our results identify the mechanistic basis governing chromosome morphogenesis in early mitosis and how distinct chromatin compaction states can be established via specific thresholds of Cdk1 kinase activity.
The orphan nuclear receptor TLX regulates neural stem cell self-renewal in the adult brain and functions primarily as a transcription repressor through recruitment of Atrophin corepressors, which bind to TLX via a conserved peptide motif termed the Atro box. Here we report crystal structures of the human and insect TLX ligand-binding domain in complex with Atro box peptides. In these structures, TLX adopts an autorepressed conformation in which its helix H12 occupies the coactivator-binding groove. Unexpectedly, H12 in this autorepressed conformation forms a novel binding pocket with residues from helix H3 that accommodates a short helix formed by the conserved ALXXLXXY motif of the Atro box. Mutations that weaken the TLX–Atrophin interaction compromise the repressive activity of TLX, demonstrating that this interaction is required for Atrophin to confer repressor activity to TLX. Moreover, the autorepressed conformation is conserved in the repressor class of orphan nuclear receptors, and mutations of corresponding residues in other members of this class of receptors diminish their repressor activities. Together, our results establish the functional conservation of the autorepressed conformation and define a key sequence motif in the Atro box that is essential for TLX-mediated repression.
All cells must sense and adapt to changing nutrient availability. However, detailed molecular mechanisms coordinating such regulatory pathways remain poorly understood. In Bacillus subtilis, nitrogen homeostasis is controlled by a unique circuitry composed of the regulator TnrA, which is deactivated by feedback-inhibited glutamine synthetase (GS) during nitrogen excess and stabilized by GlnK upon nitrogen depletion, and the repressor GlnR. Here we describe a complete molecular dissection of this network. TnrA and GlnR, the global nitrogen homeostatic transcription regulators, are revealed as founders of a new structural family of dimeric DNA-binding proteins with C-terminal, flexible, effector-binding sensors that modulate their dimerization. Remarkably, the TnrA sensor domains insert into GS intersubunit catalytic pores, destabilizing the TnrA dimer and causing an unprecedented GS dodecamer-to-tetradecamer conversion, which concomitantly deactivates GS. In contrast, each subunit of the GlnK trimer "templates" active TnrA dimers. Unlike TnrA, GlnR sensors mediate an autoinhibitory dimer-destabilizing interaction alleviated by GS, which acts as a GlnR chaperone. Thus, these studies unveil heretofore unseen mechanisms by which inducible sensor domains drive metabolic reprograming in the model Gram-positive bacterium B. subtilis.