GCN5 Maintains Muscle Integrity by Acetylating YY1 to Promote Dystrophin Expression

This work identifies a novel role for the acetyltransferase GCN5 in regulating muscle integrity through inhibition of DNA binding activity of the transcriptional repressor YY1. Here we report that in mice a muscle-specific knockout of GCN5 (Gcn5skm-/-) reduces the expression of key structural muscle proteins, including dystrophin, resulting in myopathy. Supporting our observation, a meta-analysis between the differential transcriptome of Gcn5skm-/- muscle and all available open-access data sets identified top correlations with musculoskeletal diseases in humans. GCN5 was found to acetylate YY1 at two residues (K392 and K393), which disrupts the interaction between the YY1 zinc-finger region and DNA. De/acetylation mimics for these YY1 post-translational modifications modulated muscle structural gene expression and DNA binding. Analysis of human GTEx data also found positive and negative correlations between fiber diameter and GCN5 and YY1 respectively. Collectively, our results demonstrate that GCN5 acetyltransferase activity regulates YY1 DNA binding and expression of dystrophin to modulate muscle integrity.

Roles of TOG and jelly-roll domains of centrosomal protein CEP104 in its functions in cilium elongation and Hedgehog signaling

Primary cilia are generated through the extension of the microtubule-based axoneme. Centrosomal protein 104 (CEP104) localizes to the tip of the elongating axoneme, and CEP104 mutations are linked to a ciliopathy, Joubert syndrome. Thus, CEP104 has been implicated in ciliogenesis. However, the mechanism by which CEP104 regulates ciliogenesis remains elusive. We report here that CEP104 is critical for cilium elongation but not for initiating ciliogenesis. We also demonstrated that the tumor-overexpressed gene (TOG) domain of CEP104 exhibits microtubule-polymerizing activity and that this activity is essential for the cilium-elongating activity of CEP104. Knockdown/rescue experiments showed that the N-terminal jelly-roll (JR) fold partially contributes to cilium-elongating activity of CEP104, but neither the zinc-finger region nor the SXIP motif is required for this activity. CEP104 binds to a centriole-capping protein, CP110, through the zinc-finger region and to a microtubule plus-end–binding protein, EB1, through the SXIP motif, indicating that the binding of CP110 and EB1 is dispensable for the cilium-elongating activity of CEP104. Moreover, CEP104 depletion does not affect CP110 removal from the mother centriole, which suggests that CEP104 functions after the removal of CP110. Last, we also showed that CEP104 is required for the ciliary entry of Smoothened and export of GPR161 upon Hedgehog signal activation and that the TOG domain plays a critical role in this activity. Our results define the roles of the individual domains of CEP104 in its functions in cilium elongation and Hedgehog signaling and should enhance our understanding of the mechanism underlying CEP104 mutation–associated ciliopathies.

A Unique Protection Signal in Cubitus interruptus Prevents Its Complete Proteasomal Degradation

ABSTRACT The limited proteolysis of Cubitus interruptus (Ci), the transcription factor for the developmentally and medically important Hedgehog (Hh) signaling pathway, triggers a critical switch between transcriptional repressor and activator forms. Ci repressor is formed when the C terminus of full-length Ci is degraded by the ubiquitin-proteasome pathway, an unusual reaction since the proteasome typically completely degrades its substrates. We show that several regions of Ci are required for generation of the repressor form: the zinc finger DNA binding domain, a single lysine residue (K750) near the degradation end point, and a 163-amino-acid region at the C terminus. Unlike other proteins that are partially degraded by the proteasome, dimerization is not a key feature of Ci processing. Using a pulse-chase assay in cultured Drosophila cells, we distinguish between regions required for initiation of degradation and those required for the protection of the Ci N terminus from degradation. We present a model whereby the zinc finger region and K750 together form a unique protection signal that prevents the complete degradation of Ci by the proteasome.

The Ubiquitin Ligase Triad1 Is Involved in Myelopoiesis and Interacts with the Transcriptional Repressors Gfi1 and Gfi1b.

We identified Triad1 as a gene that is upregulated by retinoic acid during the granulocytic differentiation of acute promyelocytic leukemia cells. In normal hematopoiesis, we show that Triad1 is weakly expressed in immature CD34+ bone marrow cells, and highly expressed in mature monocytes and granulocytes. Together, this suggests that Triad1 plays a role in the differentiation of hematopoietic cells. Triad1 contains a tripartite domain including two RING fingers, indicating that this protein might function as a ubiquitin E3 ligase, catalyzing the the conjugation of ubiquitin to substrate proteins thereby marking them for targeted degradation by the 26S proteasome. Using GST pull down experiments, we show that Triad1 binds to the ubiquitin conjugating (E2) enzymes UbcH6 and 7. In addition, immunoprecipitation of Triad1 in cells that were transfected with FLAG-tagged ubiquitin shows that Triad1 binds to ubiquitinated proteins, and that Triad1 is capable of self-ubiquitination, further corroborating the assumption that Triad1 acts as a E3 ubiquitin ligating enzyme. To study the role of Triad1 in hematopoiesis we overexpressed the gene in primary murine bone marrow cells using a retroviral vector that contains Triad1 in front of an IRES-GFP cassette. GFP positive cells were FACS sorted and used in colony assays (CFU-GM). Compared to empty vector controls (GFP alone), Triad1 expression resulted in more than 80% inhibition of clonogenic growth. Importantly, addition of the proteasome inhibitor MG132 (10E-8 M) reversed the Triad1-induced suppression of colony formation. Furthermore, three Triad1 expression constructs in which one of the conserved cys/his residues of the TRIAD domain (essential for function) were mutated did not show the suppressive effect on colony formation. Together, these data show that Triad1 is involved in myelopoiesis and acts through the ubiquitination of specific substrate proteins. To identify these substrates, a yeast-two-hybrid screen of a human bone marrow cDNA library was performed using the Triad1 protein as a bait. Interestingly, the transcriptional repressor Gfi1b was found to bind to Triad1. The interaction was confirmed by immunoprecipitation using GFP-Triad1 and FLAG-tagged Gfi1b transfections in mammalian cells. We show that Triad1 binds to the zinc finger region of Gfi1b. This region is very (>98%) homologous to the paralogue Gfi1. Further immunoprecipitation analyses showed that Triad1 also binds to the zinc finger region of Gfi1. Gfi1 plays an essential role in neutrophil development and Gfi1 pointmutations result in neutropenia in man. Currently, we are studying the direct ubiquitination of Gfi and Gfi1b by Triad1 in in vitro ubiquitination assays. In addition, we are studying the effect of Triad1 on the transcriptional repression of the ELA2 and other promoters by Gfi1.

The Putative Zinc Finger of the Human Cytomegalovirus IE2 86-Kilodalton Protein Is Dispensable for DNA Binding and Autorepression, Thereby Demarcating a Concise Core Domain in the C Terminus of the Protein

ABSTRACT The IE2 86-kDa gene product is an essential regulatory protein of human cytomegalovirus (HCMV) with several functions, including transactivation, negative autoregulation, and cell cycle regulation. In order to understand the physiological significance of each of the IE2 functions, discriminating mutants of IE2 are required that can be tested in a viral background. However, no such mutants of IE2 are available, possibly reflecting structural peculiarities of the large and ill-defined C-terminal domain of IE2. Here, we revisited the C-terminal domain by analyzing IE2 mutants for transactivation, DNA binding, autoregulation, and cell cycle regulation in parallel. We found it to contain an unexpectedly concise core domain (amino acids 450 to 544) that is defined by its absolute sensitivity to any kind of mutation. In contrast, the region adjacent to the core (amino acids 290 to 449) generally tolerates mutations much better. Although it contributes more specific sequence information to distinct IE2 activities, none of the mutations analyzed abolished any particular function. The core is demarcated from the adjacent region by the putative zinc finger region (amino acids 428 to 452). Surprisingly, the deletion of the putative zinc finger region from IE2 revealed that this region is entirely dispensable for any of the IE2 functions tested here in transfection assays. Our work supports the view that the 100 amino acids of the core domain hold the key to most functions of IE2. A systematic, high-density mutational analysis of this region may identify informative mutants discriminating between various IE2 functions that can then be tested in a viral background.