Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Case Reports
. 2015 Apr;47(4):338-44.
doi: 10.1038/ng.3229. Epub 2015 Mar 2.

Germline gain-of-function mutations in AFF4 cause a developmental syndrome functionally linking the super elongation complex and cohesin

Kosuke Izumi  1 , Ryuichiro Nakato  2 , Zhe Zhang  3 , Andrew C Edmondson  4 , Sarah Noon  4 , Matthew C Dulik  5 , Ramakrishnan Rajagopalan  5 , Charles P Venditti  6 , Karen Gripp  7 , Joy Samanich  8 , Elaine H Zackai  9 , Matthew A Deardorff  9 , Dinah Clark  4 , Julian L Allen  10 , Dale Dorsett  11 , Ziva Misulovin  11 , Makiko Komata  2 , Masashige Bando  2 , Maninder Kaur  4 , Yuki Katou  2 , Katsuhiko Shirahige  12 , Ian D Krantz  9
Affiliations
Case Reports

Germline gain-of-function mutations in AFF4 cause a developmental syndrome functionally linking the super elongation complex and cohesin

Kosuke Izumi et al. Nat Genet. 2015 Apr.

Abstract

Transcriptional elongation is critical for gene expression regulation during embryogenesis. The super elongation complex (SEC) governs this process by mobilizing paused RNA polymerase II (RNAP2). Using exome sequencing, we discovered missense mutations in AFF4, a core component of the SEC, in three unrelated probands with a new syndrome that phenotypically overlaps Cornelia de Lange syndrome (CdLS) that we have named CHOPS syndrome (C for cognitive impairment and coarse facies, H for heart defects, O for obesity, P for pulmonary involvement and S for short stature and skeletal dysplasia). Transcriptome and chromatin immunoprecipitation sequencing (ChIP-seq) analyses demonstrated similar alterations of genome-wide binding of AFF4, cohesin and RNAP2 in CdLS and CHOPS syndrome. Direct molecular interaction of the SEC, cohesin and RNAP2 was demonstrated. These data support a common molecular pathogenesis for CHOPS syndrome and CdLS caused by disturbance of transcriptional elongation due to alterations in genome-wide binding of AFF4 and cohesin.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Identification of novel genetic disorder and its causative gene. (a) CHOPS syndrome probands: CHOPS T254S (female with short stature, intellectual disability, chronic lung disease, obesity, brachydactyly, vertebral abnormalities, patent ductus aretriosus, horseshoe kidney and dysmorphic facial features), CHOPS T254A (male with short stature, intellectual disability, tracheomalacia, subglottic and tracheal stenosis, obesity, brachydactyly, cervical vertebrae abnormalities, ventricular septal defect and patent ductus arteriosus, cryptorchidism, hearing loss and dysmorphic facial features) and CHOPS R258W (female with short stature, intellectual disability, laryngomalacia, narrow oropharynx, brachydactyly, kyphoscoliosis, patent ductus arteriosus, ventricular septal defect, cataracts and dysmorphic facial features). Written permission to publish photograph was obtained from the parents of CHOPS syndrome probands. (b) AFF4 protein structure demonstrating the location of de novo missense mutations identified in 3 probands. Missense mutations altered highly conserved amino acid residues. NHD: N-terminal homology domain, TAD: transactivation domain, NLS: nuclear localization signal, NoLS: nucleolar localization signals, CHD: C-terminal homology domain.
Figure 2
Figure 2
Disease mechanism of CHOPS syndrome. (a) Decreased proteosomal degradation of mutant AFF4 in 293T cells. Western blot demonstrates disappearance of WT AFF4 bands with the addition of SIAH1 vector. However, such disappearance was not observed in 293T cells overexpressing mutant AFF4 vectors. The numbers beneath AFF4 bands indicate the signal intensities normalized to the band intensity of AFF4 only transfection condition of each category and alpha tubulin. (b, c) Expression level of MYC/JUN gene in patient-derived skin fibroblasts and 293T cell line with AFF4 overexpression. GM01652, GM02036 and GM08398 are control fibroblast cell lines. Elevation of MYC and JUN expression were observed in CHOPS syndrome skin fibroblast (2b) and 293T AFF4 overexpression model (2c). MYC and JUN expression was normalized against TBP. Error bars demonstrate mean ± 2 standard deviations. **P<0.01, ***P<0.001, two-tailed t-test, n= 3 per group.
Figure 3
Figure 3
Similar transcriptional profile between CHOPS syndrome and CdLS. (a) General comparison between CHOPS syndrome and CdLS demonstrated positive correlation between these two syndromes. (b) Top 250 dysregulated genes comparison. Left: Upregulated genes and downregulated genes in CdLS showed similar changes to CHOPS syndrome samples. Right: Dysregulated genes in CHOPS syndrome demonstrated similar changes to CdLS samples. The bottom of the box represents the 1st quartile, and the top of the box represents the 3rd quartile. The line in the middle of the box represents the median. The notches/whiskers demonstrate the confidence interval. (c) Volcano plots: Top left: volcano plots defining top 250 genes whose expression levels are higher (purple dots) and lower (green dots) in the CHOPs syndrome samples. These plots represent the top 250 genes that are either up- or down-regulated in the probands with the highest magnitude of difference and p < 0.05. Bottom left: Distribution of these 250 genes, whose expression is up- and down-regulated in CHOPS syndrome, in the CdLS samples. The same genes were labeled by the same colors in the bottom plot. Top right: Distribution of the top 250 genes whose expression was up- (purple dots) or down-regulated (green dots) in the CdLS syndrome samples. Bottom right: Distribution of these 250 genes, whose expression is up- or down-regulated in CdLS, in the CHOPS syndrome samples.
Figure 4
Figure 4
Western blot analysis of the SEC components in CHOPS syndrome skin fibroblast cell lines. (a) CHOPS syndrome sample cell lysates demonstrate a stronger AFF4 band, however, the amounts of NIPBL, MAU2 and SMC1 were unchanged compared to the control samples. GM02036 and GM03348 are control samples. (b) Accumulation of AFF4 in the chromatin fraction was seen in the CHOPS syndrome samples, however, the amount of ELL2 and CDK9 remained unchanged. Numbers beneath the bands represents the signal intensities normalized against the band intensity of the control GM02036 sample, and also normalized against alpha tubulin in total cellular lysates and histone H3 in chromatin fraction samples. *The asterisk indicates a nonspecific band. CdLS sample used was CDL006 with a frameshift mutation of NIPBL.
Figure 5
Figure 5
Molecular interaction among SEC, cohesin and RNAP2. Results of immunoprecipitation-Western blot. (a) Protein interaction between SEC and cohesin. STAG1 (SA1) interacts with various SEC components including AFF4, ELL2, Cyclin T1 and CDK9. Immunoprecipitation with STAG2 (SA2) did not show such interaction with SEC. Addition of DRB slightly increased the amount of SEC components interacting with SA1. (b) Protein interaction between cohesin and RNAP2. STAG1 (SA1) interacts with various forms of RNAP2. Addition of DRB (D) and flavopiridol (F) decreased the amount of RNAP2 Ser2ph and Ser5ph precipitated with STAG1. RNAP2 was not immunoprecipitated with STAG2 (SA2) antibody.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Lin C, et al. Dynamic transcriptional events in embryonic stem cells mediated by the super elongation complex (SEC). Genes Dev. 2011;25:1486–1498. - PMC - PubMed
    1. Yamaguchi Y, Shibata H, Handa H. Transcription elongation factors DSIF and NELF: promoter-proximal pausing and beyond. Biochim. Biophys. Acta. 2013;1829:98–104. - PubMed
    1. Heidemann M, Hintermair C, Voß K, Eick D. Dynamic phosphorylation patterns of RNA polymerase II CTD during transcription. Biochim. Biophys. Acta. 2013;1829:55–62. - PubMed
    1. Lin C, et al. AFF4, a component of the ELL/P-TEFb elongation complex and a shared subunit of MLL chimeras, can link transcription elongation to leukemia. Mol. Cell. 2010;37:429–437. - PMC - PubMed
    1. Liu J, Krantz ID. Cornelia de Lange syndrome, cohesin, and beyond. Clin. Genet. 2009;76:303–314. - PMC - PubMed

Publication types

MeSH terms

Associated data

-