An I47L substitution in the HOXD13 homeodomain causes a novel human limb malformation by producing a selective loss of function

Caronia, Giuliana; Goodman, Frances R.; McKeown, Carole; Scambler, Peter J.; Zappavigna, Vincenzo

doi:10.1242/dev.00396

Cited by 62 publications

(78 citation statements)

References 53 publications

(53 reference statements)

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“…The pT81-Shh reporter was described previously (3). The pSG5-HA-HOXD13, pSG5-HA-HOXD13IQN, and pGEX4T-HOXD13HD expression constructs were as described previously (4). Expression vectors for Myc-tagged Orc2 and Cdc6 were as described previously (58).…”

Section: Methodsmentioning

confidence: 99%

HOXD13 Binds DNA Replication Origins To Promote Origin Licensing and Is Inhibited by Geminin

Salsi¹,

Ferrari²,

Ferraresi

et al. 2009

Molecular and Cellular Biology

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HOX DNA-binding proteins control patterning during development by regulating processes such as cell aggregation and proliferation. Recently, a possible involvement of HOX proteins in replication origin activity was suggested by results showing that a number of HOX proteins interact with the DNA replication licensing regulator geminin and bind a characterized human origin of replication. The functional significance of these observations, however, remained unclear. We show that HOXD13, HOXD11, and HOXA13 bind in vivo all characterized human replication origins tested. We furthermore show that HOXD13 interacts with the CDC6 loading factor, promotes pre-replication complex (pre-RC) proteins assembly at origins, and stimulates DNA synthesis in an in vivo replication assay. HOXD13 expression in cultured cells accelerates DNA synthesis initiation in correlation with the earlier pre-RC recruitment onto origins during G 1 phase. Geminin, which interacts with HOXD13 as well, blocks HOXD13-mediated assembly of pre-RC proteins and inhibits HOXD13-induced DNA replication. Our results uncover a function for Hox proteins in the regulation of replication origin activity and reveal an unforeseen role for the inhibition of HOX protein activity by geminin in the context of replication origin licensing.Hox proteins belong to the large family of homeodomaincontaining DNA-binding proteins. During embryonic development they control cell fates, eventually leading to the generation of different regional identities along the primary body and limb axes (17,35). Genetic analyses, including loss-and gainof-function experiments, showed that vertebrate Hox genes modulate morphogenetic processes by controlling crucial aspects such as cell proliferation and aggregation (16). This is particularly true of 5Ј HoxA and HoxD genes, which are involved in limb patterning (reviewed in references 49 and 65). The inactivation of the Hoxd13 gene in mice, for instance, causes a phenotype resulting from defects in the proliferation and/or condensation of limb mesenchymal cells (11,14).Hox proteins have been shown to act as transcription factors, which are thought to regulate sets of target genes by binding to specific DNA sequences within the transcriptional regulatory regions of these (7,33,38,43,50,51,61,62). Recent findings, however, have hinted at a possible additional function for this family of DNA-binding proteins. Hox proteins have been found to associate with the DNA replication licensing regulator geminin (39), and a number of them have been shown to bind the human LaminB2 and other origins of replication, suggesting their possible role in origin definition and/or assembly of the replication machinery (9,13,20). The functional significance of these observations, however, remained elusive.Origins of replication are poorly defined in metazoa. A consensus sequence appears not to be required for replication initiation in human cells, and only a few origins where replication initiates from a localized site in each cell cycle have been well characterize...

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Section: Methodsmentioning

confidence: 99%

HOXD13 Binds DNA Replication Origins To Promote Origin Licensing and Is Inhibited by Geminin

Salsi¹,

Ferrari²,

Ferraresi

et al. 2009

Molecular and Cellular Biology

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“…In these cases the phenotypes can be mutation specific, as it has been reported for, e.g., MSX2, FOXC1, PITX2, or HOXD13 (Kozlowski and Walter 2000;Wilkie et al 2000;Johnson et al 2003;Saleem et al 2003). How these mutations exert their effect remains unclear, but it is likely that the mutant protein has altered binding specificity or affinity, resulting in abnormal targets or activity (Caronia et al 2003;Saleem et al 2003;Zhao et al 2007).…”

mentioning

confidence: 97%

“…Mutations in HOXD13, the most 59 gene of the HOXD cluster, include polyalanine expansions and frame-shift or missense mutations, and are associated with severe digit malformations in humans (Debeer et al 2002;Caronia et al 2003;Johnson et al 2003). Intriguingly, polyalanine expansions and frame-shift mutations are predominantly associated with the formation of extra digits and webbing between digits, collectively called synpolydactyly, while missense substitutions have been reported to cause distinctive shortening of the hands/feet (brachydactyly) (for a review, see Goodman 2002).…”

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confidence: 99%

Distinct global shifts in genomic binding profiles of limb malformation-associated HOXD13 mutations

et al. 2013

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Gene regulation by transcription factors (TFs) determines developmental programs and cell identity. Consequently, mutations in TFs can lead to dramatic phenotypes in humans by disrupting gene regulation. To date, the molecular mechanisms that actually cause these phenotypes have been difficult to address experimentally. ChIP-seq, which couples chromatin immunoprecipitation with high-throughput sequencing, allows TF function to be investigated on a genomewide scale, enabling new approaches for the investigation of gene regulation. Here, we present the application of ChIP-seq to explore the effect of missense mutations in TFs on their genome-wide binding profile. Using a retroviral expression system in chicken mesenchymal stem cells, we elucidated the mechanism underlying a novel missense mutation in HOXD13 (Q317K) associated with a complex hand and foot malformation phenotype. The mutated glutamine (Q) is conserved in most homeodomains, a notable exception being bicoid-type homeodomains that have lysine (K) at this position. Our results show that the mutation results in a shift in the binding profile of the mutant toward a bicoid/PITX1 motif. Gene expression analysis and functional assays using in vivo overexpression studies confirm that the mutation results in a partial conversion of HOXD13 into a TF with bicoid/PITX1 properties. A similar shift was not observed with another mutation, Q317R, which is associated with brachysyndactyly, suggesting that the bicoid/PITX1-shift observed for Q317K might be related to the severe clinical phenotype. The methodology described can be used to investigate a wide spectrum of TFs and mutations that have not previously been amenable to ChIP-seq experiments.

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“…43 Polyalanine coding repeat expansions in exon 1 of HOXD13 cause synpolydactyly. 44,45 Intragenic frameshift deletions, 46 missense mutations in exon 2 47,48 and an acceptor splice site mutation 49 have been associated with novel hand and/or foot malformations. It is important to note that breakpoints in chromosome 2q31 near the HOXD cluster were found in four independent patients with balanced chromosome rearrangements and various limb and skeletal malformations.…”

Section: Discussionmentioning

confidence: 99%

De novo t(12;17)(p13.3;q21.3) translocation with a breakpoint near the 5′ end of the HOXB gene cluster in a patient with developmental delay and skeletal malformations

Yue

Farcas

Thiel³

et al. 2007

Eur J Hum Genet

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A boy with severe mental retardation, funnel chest, bell-shaped thorax, and hexadactyly of both feet was found to have a balanced de novo t(12;17)(p13.3;q21.3) translocation. FISH with BAC clones and longrange PCR products assessed in the human genome sequence localized the breakpoint on chromosome 17q21.3 to a 21-kb segment that lies o30 kb upstream of the HOXB gene cluster and immediately adjacent to the 3 0 end of the TTLL6 gene. The breakpoint on chromosome 12 occurred within telomeric hexamer repeats and, therefore, is not likely to affect gene function directly. We propose that juxtaposition of the HOXB cluster to a repetitive DNA domain and/or separation from required cis-regulatory elements gave rise to a position effect.

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An I47L substitution in the HOXD13 homeodomain causes a novel human limb malformation by producing a selective loss of function

Cited by 62 publications

References 53 publications

HOXD13 Binds DNA Replication Origins To Promote Origin Licensing and Is Inhibited by Geminin

HOXD13 Binds DNA Replication Origins To Promote Origin Licensing and Is Inhibited by Geminin

Distinct global shifts in genomic binding profiles of limb malformation-associated HOXD13 mutations

De novo t(12;17)(p13.3;q21.3) translocation with a breakpoint near the 5′ end of the HOXB gene cluster in a patient with developmental delay and skeletal malformations

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