Snf2-related CREBBP Activator Protein Research Articles

GAS41 promotes H2A.Z deposition through recognition of the N terminus of histone H3 by the YEATS domain

Glioma amplified sequence 41 (GAS41), which has the Yaf9, ENL, AF9, Taf14, and Sas5 (YEATS) domain that recognizes lysine acetylation (Kac), regulates gene expression as a subunit of the SRCAP (SNF2-related CREBBP activator protein) complex that deposits histone H2A.Z at promoters in eukaryotes. The YEATS domains of the proteins AF9 and ENL recognize Kac by hydrogen bonding the aromatic cage to arginine situated just before K9ac or K27ac in the N-terminal tail of histone H3. Curiously, the YEATS domain of GAS41 binds most preferentially to the sequence that contains K14ac of H3 (H3K14ac) but lacks the corresponding arginine. Here, we biochemically and structurally elucidated the molecular mechanism by which GAS41 recognizes H3K14ac. First, stable binding of the GAS41 YEATS domain to H3K14ac required the N terminus of H3 (H3NT). Second, we revealed a pocket in the GAS41 YEATS domain responsible for the H3NT binding by crystallographic and NMR analyses. This pocket is away from the aromatic cage that recognizes Kac and is unique to GAS41 among the YEATS family. Finally, we showed that E109 of GAS41, a residue essential for the formation of the H3NT-binding pocket, was crucial for chromatin occupancy of H2A.Z and GAS41 at H2A.Z-enriched promoter regions. These data suggest that binding of GAS41 to H3NT via its YEATS domain is essential for its intracellular function.

LINC00665 sponges miR-641 to promote the progression of breast cancer by targeting the SNF2-related CREBBP activator protein (SRCAP)

ABSTRACT The regulatory network of competing endogenous RNAs (ceRNA) exists widely in tumors and affects the expression of cancer-related genes, thus playing an important role in the development and prognosis of human tumors. In this research, we explored the role and mechanism of LINC00665 as a ceRNA in breast cancer. We analyzed the expression and targets of LINC00665 in breast cancer using bioinformatics, and detected their effects on breast cancer cells by CCK8, transwell, colony formation and flow cytometry assays. From our results, LINC00665 knockdown suppressed the proliferation, migration and invasion and induced the apoptosis through inactivating the AKT/mTOR signaling pathway in MCF7 and MDA-MB-231 cells. LINC00665 had five potential downstream target miRNAs (miR-542-3p, miR-624-5p, miR-641, miR-425-5p, and miR-30-3p). In dual-luciferase report gene assay, the fluorescence activity of cells transfected with miR-641 mimics decreased, and the expression of miR-641 decreased significantly after knocking down LINC00665. miR-641 mimics significantly inhibited cell proliferation and invasion in MCF7 and MDA-MB-231 cells. We detected five potential direct targets of miR-641 using qPCR (SRCAP, SIKE1, NADK, KHDC4, and HSPG2). SRCAP expression decreased significantly in miR-641 overexpression cells and the binding of SRCAP’s 3ʹUTR and miR-641 was further confirmed by dual-luciferase report gene assay. SRCAP blocked the proliferation and invasion inhibition induced by miR-641 or si-LINC00665 in MCF7 and MDA-MB-231 cells. In conclusion, LINC00665 could promote the survival and metastasis of breast cancer cells through sponging miR-641 and targeting SRCAP. This research provided new potential targets for targeted therapy in human breast cancer.

Truncating SRCAP variants outside the Floating-Harbor syndrome locus cause a distinct neurodevelopmental disorder with a specific DNA methylation signature

SummaryTruncating variants in exons 33 and 34 of the SNF2-related CREBBP activator protein (SRCAP) gene cause the neurodevelopmental disorder (NDD) Floating-Harbor syndrome (FLHS), characterized by short stature, speech delay, and facial dysmorphism. Here, we present a cohort of 33 individuals with clinical features distinct from FLHS and truncating (mostly de novo) SRCAP variants either proximal (n = 28) or distal (n = 5) to the FLHS locus. Detailed clinical characterization of the proximal SRCAP individuals identified shared characteristics: developmental delay with or without intellectual disability, behavioral and psychiatric problems, non-specific facial features, musculoskeletal issues, and hypotonia. Because FLHS is known to be associated with a unique set of DNA methylation (DNAm) changes in blood, a DNAm signature, we investigated whether there was a distinct signature associated with our affected individuals. A machine-learning model, based on the FLHS DNAm signature, negatively classified all our tested subjects. Comparing proximal variants with typically developing controls, we identified a DNAm signature distinct from the FLHS signature. Based on the DNAm and clinical data, we refer to the condition as “non-FLHS SRCAP-related NDD.” All five distal variants classified negatively using the FLHS DNAm model while two classified positively using the proximal model. This suggests divergent pathogenicity of these variants, though clinically the distal group presented with NDD, similar to the proximal SRCAP group. In summary, for SRCAP, there is a clear relationship between variant location, DNAm profile, and clinical phenotype. These results highlight the power of combined epigenetic, molecular, and clinical studies to identify and characterize genotype-epigenotype-phenotype correlations.

A Pilot Longitudinal Evaluation of MicroRNAs for Monitoring the Cognitive Impairment in Pediatric Multiple Sclerosis

MicroRNAs (miRNAs), a class of non-coding RNAs, seem to play a key role in complex diseases like multiple sclerosis (MS), as well as in many cognitive functions associated with the disease. In a previous cross-sectional evaluation on pediatric MS (PedMS) patients, the expression of some miRNAs and their target genes were found to be associated with the scores of some neuropsychiatric tests, thus suggesting that they may be involved in early processes of cognitive impairment. To verify these data, we asked the same patients to be re-evaluated after a 1-year interval; unfortunately, only nine of them agreed to this further clinical and molecular analysis. The main results showed that 13 differentially expressed miRNAs discriminated the two time-points. Among them, the expression of miR-182-5p, miR-320a-3p, miR-744-5p and miR-192-5p significantly correlated with the attention and information processing speed performances, whereas the expression of miR-182-5p, miR-451a, miR-4742-3p and miR-320a-3p correlated with the expressive language performances. The analysis of mRNA expression uncovered 58 predicted and/or validated miRNA-target pairs, including 23 target genes, some of them already associated with cognitive impairment, such as the transducing beta like 1 X-linked receptor-1 gene (TBL1XR1), correlated to disorders of neurodevelopment; the Snf2 related CREBBP activator protein gene (SRCAP) that was found implicated in a rare form of dementia; and the glia maturation factor beta gene (GMFB), which has been reported to be implicated in neurodegeneration and neuroinflammation. No molecular pathways involving the most targeted genes survived the adjustment for multiple data. Although preliminary, these findings showed the feasibility of the methods also applied to longitudinal investigations, as well as the reliability of the obtained results. These findings should be confirmed in larger PedMS cohorts in order to identify early markers of cognitive impairment, towards which more efficient therapeutic efforts can be addressed.

Floating-Harbor Syndrome: Presentation of the First Romanian Patient with a SRCAP Mutation and Review of the Literature.

Floating-Harbor syndrome (FHS) is a rare autosomal dominant syndrome characterized by short stature with delayed bone age, retarded speech development, intellectual disability and dysmorphic facial features. Recently, dominant mutations almost exclusively clustered in the final exon of the Snf2-related CREBBP activator protein (SRCAP) gene were identified to cause FHS. Here, we report a boy with short stature, speech delay, mild intellectual disability, dysmorphic features, and with genetically confirmed FHS. To the best of our knowledge, this is the first molecularly confirmed case with this syndrome reported in Romania. An intensive program of cognitive and speech stimulation, as well as yearly neurological, psychological, ophthalmological, otorhinolaryngological, pediatric and endocrinological monitoring for our patient were designed. We propose a checklist of clinical features suggestive of FHS, based on the main clinical features, in order to facilitate the diagnosis and clinical management of this rare condition.

The first Korean case with Floating-Harbor syndrome with a novel SRCAP mutation diagnosed by targeted exome sequencing.

Floating-Harbor syndrome is a rare autosomal dominant genetic disorder associated with SRCAP mutation. To date, approximately 50 cases of Floating-Harbor syndrome have been reported, but none have been reported in Korea yet. Floating-Harbor syndrome is characterized by delayed bony maturation, unique facial features, and language impairment. Here, we present a 6-year-old boy with a triangular face, deep-set protruding eyes, low-set ears, wide nose with narrow nasal bridge, short philtrum, long thin lips, clinodactyly, and developmental delay that was transferred to our pediatric clinic for genetic evaluation. He showed progressive delay in the area of language and cognition-adaption as he grew. He had previously undergone chromosomal analysis at another hospital due to his language delay, but his karyotype was normal. We performed targeted exome sequencing, considering several syndromes with similar phenotypes. Library preparation was performed with the TruSight One sequencing panel, which enriches the sample for about 4,800 genes of clinical relevance. Massively parallel sequencing was conducted with NextSeq. An identified variant was confirmed by Sanger sequencing of the patient and his parents. Finally, the patient was confirmed as the first Korean case of Floating-Harbor syndrome with a novel SRCAP (Snf2 related CREBBP activator protein) mutation (c.7732dupT, p.Ser2578Phefs*6), resulting in early termination of the protein; it was not found in either of his healthy parents or a control population. To our knowledge, this is the first study to describe a boy with Floating-Harbor syndrome with a novel SRCAP mutation diagnosed by targeted exome sequencing in Korea.

LncKdm2b controls self-renewal of embryonic stem cells via activating expression of transcription factor Zbtb3.

Divergent long noncoding RNAs (lncRNAs) represent a major lncRNA biotype in mouse and human genomes. The biological and molecular functions of the divergent lncRNAs remain largely unknown. Here, we show that lncKdm2b, a divergent lncRNA for Kdm2b gene, is conserved among five mammalian species and highly expressed in embryonic stem cells (ESCs) and early embryos. LncKdm2b knockout impairs ESC self-renewal and causes early embryonic lethality. LncKdm2b can activate Zbtb3 by promoting the assembly and ATPase activity of Snf2-related CREBBP activator protein (SRCAP) complex in trans Zbtb3 potentiates the ESC self-renewal in a Nanog-dependent manner. Finally, Zbtb3 deficiency impairs the ESC self-renewal and early embryonic development. Therefore, our findings reveal that lncRNAs may represent an additional layer of the regulation of ESC self-renewal and early embryogenesis.

Suppression of SRCAP chromatin remodelling complex and restriction of lymphoid lineage commitment by Pcid2

Lymphoid lineage commitment is an important process in haematopoiesis, which forms the immune system to protect the host from pathogen invasion. However, how multipotent progenitors (MPP) switch into common lymphoid progenitors (CLP) or common myeloid progenitors (CMP) during this process remains elusive. Here we show that PCI domain-containing protein 2 (Pcid2) is highly expressed in MPPs. Pcid2 deletion in the haematopoietic system causes skewed lymphoid lineage specification. In MPPs, Pcid2 interacts with the Zinc finger HIT-type containing 1 (ZNHIT1) to block Snf2-related CREBBP activator protein (SRCAP) activity and prevents the deposition of histone variant H2A.Z and transcription factor PU.1 to key lymphoid fate regulator genes. Furthermore, Znhit1 deletion also abrogates H2A/H2A.Z exchange in MPPs. Thus Pcid2 controls lymphoid lineage commitment through the regulation of SRCAP remodelling activity.

Ultra-rare mutations in SRCAP segregate in Caribbean Hispanic families with Alzheimer disease.

Objective:To identify rare coding variants segregating with late-onset Alzheimer disease (LOAD) in Caribbean Hispanic families.Methods:Whole-exome sequencing (WES) was completed in 110 individuals from 31 Caribbean Hispanic families without APOE ε4 homozygous carriers. Rare coding mutations segregating in families were subsequently genotyped in additional families and in an independent cohort of Caribbean Hispanic patients and controls. SRCAP messenger RNA (mRNA) expression was assessed in whole blood from mutation carriers with LOAD, noncarriers with LOAD, and healthy elderly controls, and also from autopsied brains in 2 clinical neuropathologic cohort studies of aging and dementia.Results:Ten ultra-rare missense mutations in the Snf2-related CREBBP, activator protein (SRCAP), were found in 12 unrelated families. Compared with the frequency in Caribbean Hispanic controls and the Latino population in the Exome Aggregation Consortium, the frequency of SRCAP mutations among Caribbean Hispanic patients with LOAD was significantly enriched (p = 1.19e-16). mRNA expression of SRCAP in whole blood was significantly lower in mutation carriers with LOAD, while the expression in whole blood and in the brain was significantly higher in nonmutation carriers with LOAD. Brain expression also correlated with clinical and neuropathologic endophenotypes.Conclusions:WES in Caribbean Hispanic families with LOAD revealed ultra-rare missense mutations in SRCAP, a gene expressed in the brain and mutated in Floating-Harbor syndrome. SRCAP is a potent coactivator of the CREB-binding protein and a regulator of DNA damage response involving ATP-dependent chromatin remodeling. We hypothesize that increased expression in LOAD suggests a compensatory mechanism altered in mutation carriers.

Genome-Wide Mutational Landscape of Infant Acute Lymphoblastic Leukemia

[Background] Mixed lineage leukemia (MLL also called KMT2A) rearrangement-positive leukemia is one of the most aggressive types of leukemia. It is diagnosed predominantly in infants and typically shows a multi-lineage phenotype. Since current chemotherapy fails in more than 50% of infantile acute lymphoblastic leukemia (ALL) with MLL-rearrangement, a better understanding of biological features of the disease is important in order to develop more specific and successful treatment strategies. Despite the evident biological and clinical significance of MLL-rearrangement, it has also been shown in vitro and in vivo that MLL-rearrangement is not sufficient to induce full leukemic transformation, indicating additional hits are required for complete leukemogenesis.Recently, sequencing-based genome-wide studies have suggested collaborative involvement of RAS-PI3K pathway in infant leukemogenesis through identification of recurrent mutations affecting RAS-PI3K pathway genes, in infant ALL with or without MLL-rearrangement. However, genetic basis of infant ALL with MLL-rearrangement are not fully elucidated.[Materials and Methods] Here, we performed whole exome sequencing in leukemia samples with matched peripheral blood T lymphocyte from 46 cases of infant ALL including 3 cases with germline MLL. Our cohort included samples from the Infant Leukemia Sub-committee, Japan Children's Cancer Group. For mapping and mutation calling, we used publicly available "Genomon Pipeline".[Results] In total, 198 somatic non-silent mutations (4.3 mutations per case) were identified, including 125 missense, 4 nonsense, 8 splice site mutations, 49 frameshift indels, and 12 in-frame indels. Thirteen genes were recurrently affected, among which RAS-PI3K pathway mutations were the most frequent: KRAS (10/46 cases, 21.7%), NRAS (4/46 cases, 8.7%) and PTPN11 (3/46 cases, 6.5%). In addition, novel recurrent mutations were detected in HSP90AB1 (5/46 cases, 10.9%), MAPRE3 (2/46 cases, 4.3%) and SRCAP (2/46 cases, 4.3%), as well as known candidate driver mutations in FLT3 (7/46 cases, 15.2%), TP53 (3/46 cases, 6.5%), CHD4 (2/46 cases, 4.3%) and PAX5 (2/46 cases, 4.3%). HSP90AB1 is a member of Heat shock protein90 (Hsp90) family that encodes a group of molecular chaperones involved in stabilization and activation of multiple oncogenic proteins and pathways. Overexpression of Hsp90 is observed in ALL, chronic myeloid/lymphocytic leukemia and several non-hematological malignancies. Another recurrently mutated gene, SRCAP, encodes the core component of chromatin-remodeling Snf2-related CREBBP activator protein complex. Recurrent mutations in this gene have recently been reported in FLT3-ITD positive acute myeloid leukemia with significantly high co-occurrence of MLL3 mutations. Overall, RAS-PI3K pathway mutations were detected in 19/46 cases of infant ALL (41.3%). In our cohort, none of 3 germline MLL cases contained RAS-PI3K pathway mutations; one case had a PAX5 mutation, another had a SRCAP mutation, and the other had no candidate driver mutations. In the germline MLL case without candidate drivers, G-banding had identified a structural abnormality, t(5;15)(p15;q11.2), that is rare but reported to be predominant in infant ALL rather than other hematological/non-hematological malignancies.[Conclusion] Our results demonstrated that aberrations of cell proliferation signaling, transcription factors, as well as cell cycle/epigenetic regulators are co-operative oncogenic events in MLL-rearrangement cases. The mutation spectrum is similar to myeloid malignancies rather than lymphoid cancers. Intriguingly, we identified novel recurrent mutations in the oncogenic proteins, such as HSP90AB1, MAPRE3, and SRCAP,suggesting that these are potentially targetable by small-molecule therapy. In addition, we also illustrated the genetic differences between MLL-rearrangement and germline MLL. Thus, our mutational landscape provides a novel insight into the molecular mechanisms of infant ALL and may contribute to improving the clinical outcome of infant patients suffering from the intractable ALL. DisclosuresOgawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Expanded spectrum of exon 33 and 34 mutations in SRCAP and follow-up in patients with Floating-Harbor syndrome.

BackgroundFloating-Harbor syndrome is a rare autosomal dominant short stature syndrome with retarded speech development, intellectual disability and dysmorphic facial features. Recently dominant mutations almost exclusively located in exon 34 of the Snf2-related CREBBP activator protein gene were identified to cause FHS.MethodsHere we report the genetic analysis of 5 patients fulfilling the diagnostic criteria of FHS obtained by Sanger sequencing. All of them presented with short stature, speech delay as well as psychomotor delay and typical facial dysmorphism. Three patients showed a good response to growth hormone treatment.ResultsTwo patients demonstrate novel, heterozygous de novo frameshift mutations in exon 34 (c.7396delA and c.7218dupT) leading to premature stop mutations in SRCAP (p.Val2466Tyrfs*9 and p.Gln2407Serfs*36, respectively). In two further patients we found already known SRCAP mutations in exon 34, c.7330C > T and c.7303C > T, respectively, which also lead to premature stop codons: p.Arg2444* and p.Arg2435*. In one patient, we identified a novel de novo stop mutation in exon 33 (c.6985C > T, p.Arg2329*) demonstrating that not all FHS cases are caused by mutations in exon 34 of SRCAP.ConclusionsOur data confirm a mutational hot spot in the final exon of SRCAP in the majority of FHS patients but also show that exon 33 of this gene can be affected.

16p11.2 de novo microdeletion encompassing SRCAP gene in a patient with speech impairment, global developmental delay and behavioural problems

We describe a patient with speech impairment, global developmental delay, behavioural problems and a 186 kb de novo microdeletion on 16p11.2. There are four OMIM Phenotypes entries partially overlapping with the deleted region and related to recurrent microdeletions/microduplications in 16p11.2. A detailed review of published data shows that microdeletions/microduplications boundaries’ do not include genes that are deleted in the case here reported. The deletion encompasses 9 RefSeq genes and includes SRCAP (Snf2-related CREBBP activator protein, OMIM*611421), a disease causing gene. Recently, truncating mutations in the SRCAP gene have been shown to cause Floating-Harbor syndrome (FHS, OMIM#136140), a rare disorder characterized by peculiar facial features, short stature with delayed osseous maturation and speech impairment. The patient reported here shows few subtle phenotypic features resembling that of FHS, but she does not have sufficient signs and symptoms for the clinical diagnosis and a clinical classification based on facial gestalt is not possible. This is the first report of a 16p11.2 deletion completely removing one copy of SRCAP, suggesting that haploinsufficiency of this gene could be associated to speech impairment, global developmental delay, behavioural problems and few subtle phenotypic features resembling FHS. However, further evidence for the putative causative role of SRCAP isolated deletion is needed.

Expanded spectrum of exon 33 and 34 mutations in SRCAP and follow-up in patients with Floating-Harbor syndrome

BackgroundFloating-Harbor syndrome is a rare autosomal dominant short stature syndrome with retarded speech development, intellectual disability and dysmorphic facial features. Recently dominant mutations almost exclusively located in exon 34 of the Snf2-related CREBBP activator protein gene were identified to cause FHS.MethodsHere we report the genetic analysis of 5 patients fulfilling the diagnostic criteria of FHS obtained by Sanger sequencing. All of them presented with short stature, speech delay as well as psychomotor delay and typical facial dysmorphism. Three patients showed a good response to growth hormone treatment.ResultsTwo patients demonstrate novel, heterozygous de novo frameshift mutations in exon 34 (c.7396delA and c.7218dupT) leading to premature stop mutations in SRCAP (p.Val2466Tyrfs*9 and p.Gln2407Serfs*36, respectively). In two further patients we found already known SRCAP mutations in exon 34, c.7330C > T and c.7303C > T, respectively, which also lead to premature stop codons: p.Arg2444* and p.Arg2435*. In one patient, we identified a novel de novo stop mutation in exon 33 (c.6985C > T, p.Arg2329*) demonstrating that not all FHS cases are caused by mutations in exon 34 of SRCAP.ConclusionsOur data confirm a mutational hot spot in the final exon of SRCAP in the majority of FHS patients but also show that exon 33 of this gene can be affected.