Biallelic Variants in UBA5 Link Dysfunctional UFM1 Ubiquitin-like Modifier Pathway to Severe Infantile-Onset Encephalopathy.

The ubiquitin fold modifier 1 (UFM1) cascade is a recently identified evolutionarily conserved ubiquitin-like modification system whose function and link to human disease have remained largely uncharacterized. By using exome sequencing in Finnish individuals with severe epileptic syndromes, we identified pathogenic compound heterozygous variants in UBA5, encoding an activating enzyme for UFM1, in two unrelated families. Two additional individuals with biallelic UBA5 variants were identified from the UK-based Deciphering Developmental Disorders study and one from the Northern Finland Intellectual Disability cohort. The affected individuals (n = 9) presented in early infancy with severe irritability, followed by dystonia and stagnation of development. Furthermore, the majority of individuals display postnatal microcephaly and epilepsy and develop spasticity. The affected individuals were compound heterozygous for a missense substitution, c.1111G>A (p.Ala371Thr; allele frequency of 0.28% in Europeans), and a nonsense variant or c.164G>A that encodes an amino acid substitution p.Arg55His, but also affects splicing by facilitating exon 2 skipping, thus also being in effect a loss-of-function allele. Using an in vitro thioester formation assay and cellular analyses, we show that the p.Ala371Thr variant is hypomorphic with attenuated ability to transfer the activated UFM1 to UFC1. Finally, we show that the CNS-specific knockout of Ufm1 in mice causes neonatal death accompanied by microcephaly and apoptosis in specific neurons, further suggesting that the UFM1 system is essential for CNS development and function. Taken together, our data imply that the combination of a hypomorphic p.Ala371Thr variant in trans with a loss-of-function allele in UBA5 underlies a severe infantile-onset encephalopathy.

Biallelic UFM1 and UFC1 mutations expand the essential role of ufmylation in brain development.

The post-translational modification of proteins through the addition of UFM1, also known as ufmylation, plays a critical developmental role as revealed by studies in animal models. The recent finding that biallelic mutations in UBA5 (the E1-like enzyme for ufmylation) cause severe early-onset encephalopathy with progressive microcephaly implicates ufmylation in human brain development. More recently, a homozygous UFM1 variant was proposed as a candidate aetiology of severe early-onset encephalopathy with progressive microcephaly. Here, we establish a locus for severe early-onset encephalopathy with progressive microcephaly based on two families, and map the phenotype to a novel homozygous UFM1 mutation. This mutation has a significantly diminished capacity to form thioester intermediates with UBA5 and with UFC1 (the E2-like enzyme for ufmylation), with resulting impaired ufmylation of cellular proteins. Remarkably, in four additional families where eight children have severe early-onset encephalopathy with progressive microcephaly, we identified two biallelic UFC1 mutations, which impair UFM1-UFC1 intermediate formation with resulting widespread reduction of cellular ufmylation, a pattern similar to that observed with UFM1 mutation. The striking resemblance between UFM1- and UFC1-related clinical phenotype and biochemical derangements strongly argues for an essential role for ufmylation in human brain development. The hypomorphic nature of UFM1 and UFC1 mutations and the conspicuous depletion of biallelic null mutations in the components of this pathway in human genome databases suggest that it is necessary for embryonic survival, which is consistent with the embryonic lethal nature of knockout models for the orthologous genes.

Bcl11b SWI/SNF-complex subunit modulates intestinal adenoma and regeneration after γ-irradiation through Wnt/ß-catenin pathway.

SWI/SNF chromatin remodeling complexes constitute a highly related family of multi-subunit complexes to modulate transcription, and SWI/SNF subunit genes are collectively mutated in 20% of all human cancers. Bcl11b is a SWI/SNF subunit and acts as a haploinsufficient tumor suppressor in leukemia/lymphomas. Here, we show expression of Bcl11b in intestinal crypt cells and promotion of intestinal tumorigenesis by Bcl11b attenuation in Apc (min/+) mice. Of importance, mutations or allelic loss of BCL11B was detected in one-third of human colon cancers. We also show that attenuated Bcl11b activity in the crypt base columnar (CBC) cells expressing the Lgr5 stem cell marker enhanced regeneration of intestinal epithelial cells after the radiation-induced injury. Interestingly, BCL11B introduction in human cell lines downregulated transcription of ß-catenin target genes, whereas Bcl11b attenuation in Lgr5(+) CBCs increased expression of ß-catenin targets including c-Myc and cyclin D1. Together, our results argue that Bcl11b impairment promotes tumor development in mouse and human intestine at least in part through deregulation of ß-catenin pathway.

Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T-cell malignancy. HTLV-1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T-cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV-1-infected T-cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV-1-infected T-cells. Lentiviral transduction of Tax in a human T-cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF-κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV-2 Tax2 protein reduced the BCL11B protein expression in T-cells. Seven HTLV-1-infected T-cell lines, including three ATL-derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T-cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV-1-infected T-cells; Tax-mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV-1-infected T-cells.

Successful management of severe intrahepatic cholestasis of pregnancy: report of a first Japanese case.

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) is a cholestasis condition caused by elevated levels of serum bile acids that mainly occurs in the third trimester of pregnancy. Maternal symptoms include pruritus; elevation of transaminases, biliary enzymes, and bilirubin levels; and abnormal liver function tests. Fetal symptoms include spontaneous preterm labor, fetal distress, and intrauterine death. It is more prevalent in the Caucasians and is rarely found in Asian countries, including Japan. The etiology of ICP has been reported as involving various factors such as, environmental factors, hormone balance, and genetic components. The genetic factors include single-nucleotide polymorphisms (SNPs) in the genes of canalicular transporters, including ABCB4 and ABCB11. It has also been reported that the combination of these SNPs induces severe cholestasis and liver dysfunction. CASE PRESENTATION: Here, we report for the first time a 24-year Japanese case of severe ICP diagnosed by typical symptoms, serum biochemical analysis, and treated with the administration of ursodeoxycholic acid which improved cholestasis and liver injury and prevented fetal death. The sequence analysis showed SNPs reported their association with ICP in the ABCB11 (rs2287622, V444A) and ABCB4 (rs1202283, N168N) loci. CONCLUSION: The risk of ICP has been reported to be population-specific, and it is rare in the Japanese population. Our case was successfully treated with ursodeoxycholic acid and the genetic sequence analysis has supported the diagnosis. Because genetic variation in ABCB4 and ABCB11 has also been reported in the Japanese population, we need to be aware of potential ICP cases in pregnant Japanese women although further studies are necessary.

Cell of origin in radiation-induced premalignant thymocytes with differentiation capability in mice conditionally losing one Bcl11b allele.

Bcl11b is a haploinsufficient tumor suppressor, mutations or deletion of which has been found in 10-16% of T-cell acute lymphoblastic leukemias. Bcl11b(KO) (/+) heterozygous mice are susceptible to thymic lymphomas, a model of T-cell acute lymphoblastic leukemia, when γ-irradiated, and irradiated Bcl11b(KO) (/+) mice generate clonally expanding or premalignant thymocytes before thymic lymphoma development. Cells with radiation-induced DNA damages are assumed to be the cells of origin in tumors; however, which thymocyte is the tumor cell origin remains obscure. In this study we generated Bcl11b(flox/+) ;Lck-Cre and Bcl11b(flox/+) ;CD4-Cre mice; in the former, loss of one Bcl11b allele occurs in thymocytes at the immature CD4(-) CD8(-) stage, whereas in the latter the loss occurs in the more differentiated CD4(+) CD8(+) double-positive stage. We examined clonal expansion and differentiation of thymocytes in mice 60 days after 3 Gy γ-irradiation. Half (9/18) of the thymuses in the Bcl11b(flox/+) ;Lck-Cre group showed limited rearrangement sites at the T-cell receptor-ß (TCRß) locus, indicating clonal cell expansion, but none in the Bcl11b(flox/+) ;CD4-Cre group did. This indicates that the origin of the premalignant thymocytes is not in double-positive cells but immature thymocytes. Interestingly, those premalignant thymocytes underwent rearrangement at various different sites of the TCRα locus and the majority showed a higher expression of TCRß and CD8, and more differentiated phenotypes. This suggests the existence of a subpopulation of immature cells within the premalignant cells that is capable of proliferating and continuously producing differentiated thymocytes.

An atypical LIR motif within UBA5 (ubiquitin like modifier activating enzyme 5) interacts with GABARAP proteins and mediates membrane localization of UBA5.

Short linear motifs, known as LC3-interacting regions (LIRs), interact with mactoautophagy/autophagy modifiers (Atg8/LC3/GABARAP proteins) via a conserved universal mechanism. Typically, this includes the occupancy of 2 hydrophobic pockets on the surface of Atg8-family proteins by 2 specific aromatic and hydrophobic residues within the LIR motifs. Here, we describe an alternative mechanism of Atg8-family protein interaction with the non-canonical UBA5 LIR, an E1-like enzyme of the ufmylation pathway that preferentially interacts with GABARAP but not LC3 proteins. By solving the structures of both GABARAP and GABARAPL2 in complex with the UBA5 LIR, we show that in addition to the binding to the 2 canonical hydrophobic pockets (HP1 and HP2), a conserved tryptophan residue N-terminal of the LIR core sequence binds into a novel hydrophobic pocket on the surface of GABARAP proteins, which we term HP0. This mode of action is unique for UBA5 and accompanied by large rearrangements of key residues including the side chains of the gate-keeping K46 and the adjacent K/R47 in GABARAP proteins. Swapping mutations in LC3B and GABARAPL2 revealed that K/R47 is the key residue in the specific binding of GABARAP proteins to UBA5, with synergetic contributions of the composition and dynamics of the loop L3. Finally, we elucidate the physiological relevance of the interaction and show that GABARAP proteins regulate the localization and function of UBA5 on the endoplasmic reticulum membrane in a lipidation-independent manner.Abbreviations: ATG: AuTophaGy-related; EGFP: enhanced green fluorescent protein; GABARAP: GABA-type A receptor-associated protein; ITC: isothermal titration calorimetry; KO: knockout; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NMR: nuclear magnetic resonance; RMSD: root-mean-square deviation of atomic positions; TKO: triple knockout; UBA5: ubiquitin like modifier activating enzyme 5.

Autophagy regulates lipid metabolism through selective turnover of NCoR1.

Selective autophagy ensures the removal of specific soluble proteins, protein aggregates, damaged mitochondria, and invasive bacteria from cells. Defective autophagy has been directly linked to metabolic disorders. However how selective autophagy regulates metabolism remains largely uncharacterized. Here we show that a deficiency in selective autophagy is associated with suppression of lipid oxidation. Hepatic loss of Atg7 or Atg5 significantly impairs the production of ketone bodies upon fasting, due to decreased expression of enzymes involved in ß-oxidation following suppression of transactivation by PPARα. Mechanistically, nuclear receptor co-repressor 1 (NCoR1), which interacts with PPARα to suppress its transactivation, binds to the autophagosomal GABARAP family proteins and is degraded by autophagy. Consequently, loss of autophagy causes accumulation of NCoR1, suppressing PPARα activity and resulting in impaired lipid oxidation. These results suggest that autophagy contributes to PPARα activation upon fasting by promoting degradation of NCoR1 and thus regulates ß-oxidation and ketone bodies production.

Negative Regulation of the Keap1-Nrf2 Pathway by a p62/Sqstm1 Splicing Variant.

A key antioxidant pathway, the Keap1-Nrf2 system, is regulated by p62/Sqstm1 via multiple mechanisms, including gene expression, posttranslational modifications (such as ubiquitination and phosphorylation), and autophagic degradation of p62/Sqstm1 and Keap1. Here we demonstrate a novel mode of regulation of the Keap1-Nrf2 system, mediated by a splicing variant of p62/Sqstm1 pre-mRNA. Ensembl database searches and subsequent biochemical analyses of mice revealed the presence of an mRNA that encodes a p62/Sqstm1 protein lacking the Keap1-interacting region (KIR), which is essential for the interaction with Keap1. Like full-length p62, the variant was induced under conditions in which Nrf2 was activated (e.g., impairment of autophagy), formed oligomers with itself and/or the full-length protein, and was degraded by autophagy. However, the variant failed to interact with Keap1 and sequester it in variant-positive aggregates. Remarkably, while full-length p62 stabilized Nrf2 and induced the gene expression of Nrf2 targets, the variant increased the amount of Keap1 and enhanced ubiquitination of Nrf2, thereby suppressing the induction of Nrf2 targets. Hepatocytes isolated from genetically modified mice that express full-length p62, but not the variant, were susceptible to activation of Nrf2 in response to stress. Collectively, our results suggest that splicing of p62/Sqstm1 pre-mRNA negatively regulates the Keap1-Nrf2 pathway.

PKM1 Confers Metabolic Advantages and Promotes Cell-Autonomous Tumor Cell Growth.

Expression of PKM2, which diverts glucose-derived carbon from catabolic to biosynthetic pathways, is a hallmark of cancer. However, PKM2 function in tumorigenesis remains controversial. Here, we show that, when expressed rather than PKM2, the PKM isoform PKM1 exhibits a tumor-promoting function in KRASG12D-induced or carcinogen-initiated mouse models or in some human cancers. Analysis of Pkm mutant mouse lines expressing specific PKM isoforms established that PKM1 boosts tumor growth cell intrinsically. PKM1 activated glucose catabolism and stimulated autophagy/mitophagy, favoring malignancy. Importantly, we observed that pulmonary neuroendocrine tumors (NETs), including small-cell lung cancer (SCLC), express PKM1, and that PKM1 expression is required for SCLC cell proliferation. Our findings provide a rationale for targeting PKM1 therapeutically in certain cancer subtypes, including pulmonary NETs.

A novel approach to assess the ubiquitin-fold modifier 1-system in cells.

The ubiquitin-fold modifier 1 (UFM1)-system, a ubiquitin-like protein conjugation system, is involved in the development of breast cancer and several hereditary neurological syndromes. However, the molecular mechanisms of UFM1-related pathogenesis remain unclear. Here, we show that in the absence of UFSP2, a deconjugating enzyme for UFM1, ectopic expression of both UFL1 and UFBP1, which serve as the E3-ligase complex for the UFM1-system, dramatically increases UFM1-conjugate formation at the endoplasmic reticulum. Utilizing this system, we were able to attribute disease-related isoforms of UBA5, the E1 enzyme for UFM1, to decreased UFM1-conjugate formation. Our procedure allows the assessment of UFM1-conjugate formation in cells and the identification of UFM1-targets, both of which are needed to clarify the pathophysiological role of the UFM1-system.

Predisposition to mouse thymic lymphomas in response to ionizing radiation depends on variant alleles encoding metal-responsive transcription factor-1 (Mtf-1).

Genetic predisposition to cancers is significant to public health because a high proportion of cancers probably arise in a susceptible human subpopulation. Using a mouse model of gamma-ray-induced thymic lymphomas, we performed linkage analysis and haplotype mapping that suggested Mtf-1, metal-responsive transcription factor-1 (Mtf-1), as a candidate lymphoma susceptibility gene. Sequence analysis revealed a polymorphism of Mtf-1 that alters the corresponding amino acid at position 424 in the proline-rich domain from a serine in susceptibility strains to proline in resistant strains. The transcriptional activity of Mtf-1 encoding serine and proline was compared by transfecting the DNA to Mtf-1-null cells, and the change to proline conferred a higher metal responsiveness in transfections. Furthermore, the resistant congenic strains possessing the Mtf-1 allele of proline type exhibited higher radiation inducibility of target genes than susceptible background strains having the Mtf-1 allele of serine type. Since products of the targets such as metallothionein are able to suppress cellular stresses generated by irradiation, these results suggest that highly inducible strains having Mtf-1 of proline type are refractory to radiation effects and hence are resistant to lymphoma development.

p62/Sqstm1 promotes malignancy of HCV-positive hepatocellular carcinoma through Nrf2-dependent metabolic reprogramming.

p62/Sqstm1 is a multifunctional protein involved in cell survival, growth and death, that is degraded by autophagy. Amplification of the p62/Sqstm1 gene, and aberrant accumulation and phosphorylation of p62/Sqstm1, have been implicated in tumour development. Herein, we reveal the molecular mechanism of p62/Sqstm1-dependent malignant progression, and suggest that molecular targeting of p62/Sqstm1 represents a potential chemotherapeutic approach against hepatocellular carcinoma (HCC). Phosphorylation of p62/Sqstm1 at Ser349 directs glucose to the glucuronate pathway, and glutamine towards glutathione synthesis through activation of the transcription factor Nrf2. These changes provide HCC cells with tolerance to anti-cancer drugs and proliferation potency. Phosphorylated p62/Sqstm1 accumulates in tumour regions positive for hepatitis C virus (HCV). An inhibitor of phosphorylated p62-dependent Nrf2 activation suppresses the proliferation and anticancer agent tolerance of HCC. Our data indicate that this Nrf2 inhibitor could be used to make cancer cells less resistant to anticancer drugs, especially in HCV-positive HCC patients.

Bcl11b transcription factor plays a role in the maintenance of the ameloblast-progenitors in mouse adult maxillary incisors.

Rodent incisors maintain the ability to grow continuously and their labial dentin is covered with enamel. Bcl11b zinc-finger transcription factor is expressed in ameloblast progenitors in mouse incisors and its absence in Bcl11b(KO/KO) mice results in a defect in embryonic tooth development. However, the role of Bcl11b in incisor maintenance in adult tissue was not studied because of death at birth in Bcl11b(KO/KO) mice. Here, we examined compound heterozygous Bcl11b(S826G/KO) mice, one allele of which has an amino acid substitution of serine at position 826 for glycine, that exhibited hypoplastic maxillary incisors with lower concentrations of minerals at the enamel and the dentin, accompanying the maxillary bone hypoplasia. Histological examinations revealed hypoplasia of the labial cervical loop in incisors, shortening of the ameloblast progenitor region, and impairment in differentiation and proliferation of ameloblast-lineage cells. Interestingly, however, juvenile mice at 5days after birth did not show marked change in these phenotypes. These results suggest that attenuated Bcl11b activity impairs ameloblast progenitors and incisor maintenance. The number of BrdU label-retaining cells, putative stem cells, was lower in Bcl11b(S826G/KO) incisors, which suggests the incisor hypoplasia may be in part a result of the decreased number of stem cells. Interestingly, the level of Shh and FGF3 expressions, which are assumed to play key roles in the development and maintenance of ameloblasts and odontoblasts, was not decreased, though the expressed areas were more restricted in ameloblast progenitor and mesenchyme regions of Bcl11b(S826G/KO) incisors, respectively. Those data suggest that the incisor maintenance by Bcl11b is not directly related to the FGF epithelial-mesenchymal signaling loop including Shh but is intrinsic to ameloblast progenitors and possibly stem cells.

BCL11B tumor suppressor inhibits HDM2 expression in a p53-dependent manner.

BCL11B is a C2H2 zinc finger transcription factor that acts as a haploinsufficient tumor suppressor. Mutations and deletion in the human orthologue BCL11B have been identified in human T-cell acute lymphoblastic leukemia (T-ALL) and a mouse model of thymic lymphomas. Bcl11b(KO/+)p53(KO/+) doubly heterozygous mice, but not Bcl11b(KO/+) heterozygous mice, spontaneously develop thymic lymphomas at a high frequency, suggesting cooperativity of BCL11B and p53 in cancer development. In this study, we have examined whether or not BCL11B directly affects the p53 signaling pathway including HDM2, a ubiquitin ligase for p53 degradation. The p53 pathway regulates cell proliferation and the response to DNA damages to maintain genome integrity. Here we show that BCL11B binds to human HDM2-P2 promoter by ChIP (chromatin immuno-precipitation) assay and inhibits HDM2 expression in a p53-dependent manner. Deletion of the distal p53 responsive element in HDM2 promoter region or the lack of p53 in HCT116 cells greatly reduced the repressive effect of BCL11B on HDM2-P2 promoter activity. The repressive activity was alleviated in γ-ray induced DNA damage conditions that activate p53, suggesting interaction between BCL11B and p53 for HDM2 expression. These date suggest that BCL11B affects the activity of the p53-HDM2 feedback loop in basal and irradiated conditions. This may be a mechanism underlying the leukemic transformation in T-ALL and in Bcl11b(KO/+)p53(KO/+) mouse thymocytes.

Clonally expanding thymocytes having lineage capability in gamma-ray-induced mouse atrophic thymus.

PURPOSE: To characterize, in the setting of gamma-ray-induced atrophic thymus, probable prelymphoma cells showing clonal growth and changes in signaling, including DNA damage checkpoint. METHODS AND MATERIALS: A total of 111 and 45 mouse atrophic thymuses at 40 and 80 days, respectively, after gamma-irradiation were analyzed with polymerase chain reaction for D-J rearrangements at the TCRbeta locus, flow cytometry for cell cycle, and Western blotting for the activation of DNA damage checkpoints. RESULTS: Limited D-J rearrangement patterns distinct from normal thymus were detected at high frequencies (43 of 111 for 40-day thymus and 21 of 45 for 80-day thymus). Those clonally expanded thymocytes mostly consisted of CD4(+)CD8(+) double-positive cells, indicating the retention of lineage capability. They exhibited pausing at a late G1 phase of cell cycle progression but did not show the activation of DNA damage checkpoints such as gammaH2AX, Chk1/2, or p53. Of interest is that 17 of the 52 thymuses showing normal D-J rearrangement patterns at 40 days after irradiation showed allelic loss at the Bcl11b tumor suppressor locus, also indicating clonal expansion. CONCLUSION: The thymocytes of clonal growth detected resemble human chronic myeloid leukemia in possessing self-renewal and lineage capability, and therefore they can be a candidate of the lymphoma-initiating cells.

Two distinct methods analyzing chromatin structure using centrifugation and antibodies to modified histone H3: both provide similar chromatin states of the Rit1/Bcl11b gene.

Chromatin state of a 2-Mb region harboring Rit1/Bcl11b on mouse chromosome 12 was examined using two distinct methods. One is ChIP assay examining the degree of enrichment with histone H3 methylated at lysine 9 (H3-mLys9) in chromatin and the other is H/E (heterochromatin/euchromatin) assay that measures a chromatin condensation state by using centrifugation. The ChIP assay showed that a 50-kb interval covering the gene and an upstream region constituted chromatin enriched with unmethylated H3-mLys9 in cells expressing Rit1 compared to cells not expressing Rit1. In contrast, regions other than the 50-kb interval did not show much difference in the enrichment between the two different types of cells. On the other hand, H/E assay of two expressing and two non-expressing tissues provided compatible fractionation patterns, suggesting that the chromatin condensation state detected by H/E assay is correlated with the chromatin state controlled by histone H3 tail modification linked to gene expression. These results indicate that the centrifugation-based H/E assay should provide a new approach to the regulation of chromatin structure with respect to its condensation state, complementing ChIP assays.