Mutational scanning ofCRXclassifies clinical variants and reveals biochemical properties of the transcriptional effector domain
James L. ShepherdsonDavid M. GranasJie LiZara ShariffStephen P. PlassmeyerAlex S. HolehouseMichael A. WhiteBarak A. Cohen
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Abstract Cone-Rod Homeobox, encoded by CRX , is a transcription factor (TF) essential for the terminal differentiation and maintenance of mammalian photoreceptors. Structurally, CRX comprises an ordered DNA-binding homeodomain and an intrinsically disordered transcriptional effector domain. Although a handful of human variants in CRX have been shown to cause several different degenerative retinopathies with varying cone and rod predominance, as with most human disease genes the vast majority of observed CRX genetic variants are uncharacterized variants of uncertain significance (VUS). We performed a deep mutational scan (DMS) of nearly all possible single amino acid substitution variants in CRX, using an engineered cell-based transcriptional reporter assay. We measured the ability of each CRX missense variant to transactivate a synthetic fluorescent reporter construct in a pooled fluorescence-activated cell sorting assay and compared the activation strength of each variant to that of wild-type CRX to compute an activity score, identifying thousands of variants with altered transcriptional activity. We calculated a statistical confidence for each activity score derived from multiple independent measurements of each variant marked by unique sequence barcodes, curating a high-confidence list of nearly 2,000 variants with significantly altered transcriptional activity compared to wild-type CRX. We evaluated the performance of the DMS assay as a clinical variant classification tool using gold-standard classified human variants from ClinVar, and determined that activity scores could be used to identify pathogenic variants with high specificity. That this performance could be achieved using a synthetic reporter assay in a foreign cell type, even for a highly cell type-specific TF like CRX, suggests that this approach shows promise for DMS of other TFs that function in cell types that are not easily accessible. Per-position average activity scores closely aligned to a predicted structure of the ordered homeodomain and demonstrated position-specific residue requirements. The intrinsically disordered transcriptional effector domain, by contrast, displayed a qualitatively different pattern of substitution effects, following compositional constraints without specific residue position requirements in the peptide chain. The observed compositional constraints of the effector domain were consistent with the acidic exposure model of transcriptional activation. Together, the results of the CRX DMS identify molecular features of the CRX effector domain and demonstrate clinical utility for variant classification.Homeobox genes define a class of genes containing homeobox sequences and encoding proteins, that are identified as transcription regulator controlling cellular development and differentiation, and controlling patterns of gene expression. LIM homeobox genes contain both a homeobox and a conservative sequences (encoded LIM domain). The members of the LIM homeobox genes family which have recently been identified include lin-11, mec-3, apterous(ap), isl-1, LH-2, Rlim, lim-1, lmx-1, Xlim-1 and Xlim-3. Recent findings suggested that products expressed by LIM homeobox genes family play an important role in adjusting differentiation and developmental of the organism. The effects of LIM homeobox genes in the differentiation and development of neurons are currently attracting more and more attention.
Homeobox A1
Homeobox protein Nkx-2.5
HNF1B
DLX5
EMX2
LIM domain
CDX2
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Homeobox A1
Homeobox protein Nkx-2.5
HNF1B
CDX2
EMX2
DLX5
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There was a highly conservative homeobox in nearly all homeobox gene family,which used for encoding transcription regulatory factors(homeobox protein) and generated the accurate regulating action in cells proliferation and differentiation.The mutation of homeobox gene not only resulted in development malformation,but also had a close relationship with the occurrence and development of some tumours.The structural features of homeobox gene and the effect in tumours were summarized.
Homeobox A1
Homeobox protein Nkx-2.5
HNF1B
EMX2
DLX5
CDX2
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The dentition is a segmental system whose evolution and morphology bears analogy to the evolution of segmentation in the vertebral column and limb. Combinatorial expression of members of the large "Hox" class of homeobox regulatory genes has been shown to play an important role in positional specification in these skeletal systems. This raises the possibility that homeobox genes are also used for positional specification in the dentition, and several homeobox genes are known to be expressed in developing teeth. To identify additional dentally expressed homeobox genes, cDNA from from murine tooth germs at 9.5, 14.5, and 17.5 days gestational age was amplified by PCR using sets of degenerate primers to the homeodomains of 18 different classes of homeobox genes. Amplification products were cloned and sequenced and compared to known gene sequences. To date this approach has confirmed the presence of Msx1, Msx2, Dlx1, and Dlx2, and identified several other homeobox genes not previously known to be expressed in teeth: Dbx, MHox, and Mox2A, plus an additional Dlx gene, Dlx7. The Msx and Dlx genes are the best current candidates for a combinatorial mechanism that controls the differentiation of structures within and between teeth, and perhaps also the evolution of those structures.
Homeobox protein Nkx-2.5
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Abstract Over the past 25 years, numerous homeobox genes have been identified and their functions have been elucidated. Many studies in various plant species have revealed that Knotted‐like homeobox ( KNOX ) genes, the first identified homeobox gene in plants, play a profound role in shoot apical meristem organisation, maintenance, and leaf initiation. In addition to the KNOX gene, numerous other homeobox genes have been discovered (e.g., BEL1‐like homeodomain , Homeodomain leucine zipper , and WUSCHEL‐related homeobox genes) and play important roles in plant development. Moreover, in recent years, it has become clear that the homeobox genes also contribute to morphological diversity in plants. Key Concepts Homeobox (HB) genes encode a subset of transcription factors that contain a homeodomain In plants, several groups of HB genes (e.g., KNOTTED‐like homeobox , BEL1‐like homeodomain , Homeodomain leucine zipper , and WUSCHEL‐related homeobox genes) have been shown to have a distinct role in the development. The HB genes are involved in a wide range of developmental processes (e.g., stem cell maintenance in the SAM and floral meristems, cambium, and root meristem, leaf development, vascular development, embryo patterning, etc.). Recent studies revealed that changes in HB genes led to leaf form diversification and that HB genes are involved in alteration in response to environmental changes in a single plant.
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EMX2
Homeobox protein Nkx-2.5
HNF1B
DLX5
CDX2
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Proteome
Identification
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Homeobox transcription factors are involved in various aspects of plant development, including maintenance of the biosynthesis and signaling pathways of different hormones. However, few direct targets of homeobox proteins have been identified. We here show that overexpression of rice homeobox gene HOX1a resulted in enhanced gibberellin (GA) response, indicating a positive effect of HOX1a in GA signaling. HOX1a is induced by GA and encodes a homeobox transcription factor with transcription repression activity. In addition, HOX1a suppresses the transcription of early flowering1 (EL1), a negative regulator of GA signaling, and further electrophoretic mobility shift assay and chromatin immunoprecipitation analysis revealed that HOX1a directly bound to the promoter region of EL1 to suppress its expression and stimulate GA signaling. These results demonstrate that HOX1a functions as a positive regulator of GA signaling by suppressing EL1, providing informative hints on the study of GA signaling.
Chromatin immunoprecipitation
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The goldfish (Carassius auratus) visual pathway displays continuous growth and plasticity throughout life. Since homeobox genes are important transcriptional regulators in development, we searched for homeobox genes in the adult goldfish retina and brain. Using the PCR, we discovered a repertoire of homeobox sequences expressed in these tissues. In addition to isolating homeodomain sequences found in the vertebrate Hox gene clusters, a sequence identical to the chicken CHox7 homeodomain was characterized. Furthermore, a sequence with significant homologies to the Xenopus XIHbox8 and leech Htr-A2 homeodomains was identified, and these sequences may define an additional class of homeodomain. Finally, a sequence belonging to the paired class (prd) of homeodomains is reported. Homeobox gene expression in the adult goldfish retina and brain may be associated with the persistent developmental features of these tissues.
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EMX2
Homeobox protein Nkx-2.5
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Homeobox protein Nkx-2.5
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