The IGH locus relocalizes to a "recombination compartment" in the perinucleolar region of differentiating B-lymphocytes.

The immunoglobulin heavy chain (IGH) gene loci are subject to specific recombination events during B-cell differentiation including somatic hypermutation and class switch recombination which mark the end of immunoglobulin gene maturation in germinal centers of secondary lymph nodes. These two events rely on the activity of activation-induced cytidine deaminase (AID) which requires DNA double strand breaks be created, a potential danger to the cell. Applying 3D-fluorescence in situ hybridization coupled with immunofluorescence staining to a previously described experimental system recapitulating normal B-cell differentiation ex vivo, we have kinetically analyzed the radial positioning of the two IGH gene loci as well as their proximity with the nucleolus, heterochromatin and γH2AX foci. Our observations are consistent with the proposal that these IGH gene rearrangements take place in a specific perinucleolar "recombination compartment" where AID could be sequestered thus limiting the extent of its potentially deleterious off-target effects.

Mislocalization of centromeric histone H3 variant CENP-A contributes to chromosomal instability (CIN) in human cells.

Chromosomal instability (CIN) is a hallmark of many cancers and a major contributor to tumorigenesis. Centromere and kinetochore associated proteins such as the evolutionarily conserved centromeric histone H3 variant CENP-A, associate with centromeric DNA for centromere function and chromosomal stability. Stringent regulation of cellular CENP-A levels prevents its mislocalization in yeast and flies to maintain genome stability. CENP-A overexpression and mislocalization are observed in several cancers and reported to be associated with increased invasiveness and poor prognosis. We examined whether there is a direct relationship between mislocalization of overexpressed CENP-A and CIN using HeLa and chromosomally stable diploid RPE1 cell lines as model systems. Our results show that mislocalization of overexpressed CENP-A to chromosome arms leads to chromosome congression defects, lagging chromosomes, micronuclei formation and a delay in mitotic exit. CENP-A overexpressing cells showed altered localization of centromere and kinetochore associated proteins such as CENP-C, CENP-T and Nuf2 leading to weakened native kinetochores as shown by reduced interkinetochore distance and CIN. Importantly, our results show that mislocalization of CENP-A to chromosome arms is one of the major contributors for CIN as depletion of histone chaperone DAXX prevents CENP-A mislocalization and rescues the reduced interkinetochore distance and CIN phenotype in CENP-A overexpressing cells. In summary, our results establish that CENP-A overexpression and mislocalization result in a CIN phenotype in human cells. This study provides insights into how overexpression of CENP-A may contribute to CIN in cancers and underscore the importance of understanding the pathways that prevent CENP-A mislocalization for genome stability.

Potential biomarkers of DNA replication stress in cancer.

Oncogene activation is an established driver of tumorigenesis. An apparently inevitable consequence of oncogene activation is the generation of DNA replication stress (RS), a feature common to most cancer cells. RS, in turn, is a causal factor in the development of chromosome instability (CIN), a near universal feature of solid tumors. It is likely that CIN and RS are mutually reinforcing drivers that not only accelerate tumorigenesis, but also permit cancer cells to adapt to diverse and hostile environments. This article reviews the genetic changes present in cancer cells that influence oncogene-induced RS and CIN, with a particular emphasis on regions of the human genome that show enhanced sensitivity to the destabilizing effects of RS, such as common fragile sites. Because RS exists in a wide range of cancer types, we propose that the proteins involved counteracting this stress are potential biomarkers for indicating the degree of RS in cancer specimens. To test this hypothesis, we conducted a pilot study to validate whether some of proteins that are known from in vitro studies to play an essential role in the RS pathway could be suitable as a biomarker. Our results indicated that this is possible. With this review and pilot study, we aim to accelerate the development of a biomarker for analysis of RS in tumor biopsy specimens, which could ultimately help to stratify patients for different forms of therapy such as the RS inhibitors already undergoing clinical trials.

BMPR1B mutation causes Pierre Robin sequence.

BACKGROUND: We investigated a large family with Pierre Robin sequence (PRS). AIM OF THE STUDY: This study aims to determine the genetic cause of PRS. RESULTS: The reciprocal translocation t(4;6)(q22;p21) was identified to be segregated with PRS in a three-generation family. Whole-genome sequencing and Sanger sequencing successfully detected breakpoints in the intragenic regions of BMRP1B and GRM4. We hypothesized that PRS in this family was caused by (i) haploinsufficiency for BMPR1B or (ii) a gain of function mechanism mediated by the BMPR1B-GRM4 fusion gene. In an unrelated family, we identified another BMPR1B-splicing mutation that co-segregated with PRS. CONCLUSION: We detected two BMPR1B mutations in two unrelated PRS families, suggesting that BMPR1B disruption is probably a cause of human PRS. METHODS: GTG banding, comparative genomic hybridization, whole-genome sequencing, and Sanger sequencing were performed to identify the gene causing PRS.

Estimating the effects of transcription factors binding and histone modifications on gene expression levels in human cells.

Transcription factors and histone modifications are vital for the regulation of gene expression. Hence, to estimate the effects of transcription factors binding and histone modifications on gene expression, we construct a statistical model for the genome-wide 15 transcription factors binding data, 10 histone modifications profiles and DNase-I hypersensitivity data in three mammalian. Remarkably, our results show POLR2A and H3K36me3 can highly and consistently predict gene expression in three cell lines. And H3K4me3, H3K27me3 and H3K9ac are more reliable predictors than other histone modifications in human embryonic stem cells. Moreover, genome-wide statistical redundancies exist within and between transcription factors and histone modifications, and these phenomena may be caused by the regulation mechanism. In further study, we find that even though transcription factors and histone modifications offer similar effects on expression levels of genome-wide genes, the effects of transcription factors and histone modifications on predictive abilities are different for genes in independent biological processes.

Translin and Trax differentially regulate telomere-associated transcript homeostasis.

Translin and Trax proteins are highly conserved nucleic acid binding proteins that have been implicated in RNA regulation in a range of biological processes including tRNA processing, RNA interference, microRNA degradation during oncogenesis, spermatogenesis and neuronal regulation. Here, we explore the function of this paralogue pair of proteins in the fission yeast. Using transcript analysis we demonstrate a reciprocal mechanism for control of telomere-associated transcripts. Mutation of tfx1+ (Trax) elevates transcript levels from silenced sub-telomeric regions of the genome, but not other silenced regions, such as the peri-centromeric heterochromatin. In the case of some sub-telomeric transcripts, but not all, this elevation is dependent on the Trax paralogue, Tsn1 (Translin). In a reciprocal fashion, Tsn1 (Translin) serves to repress levels of transcripts (TERRAs) from the telomeric repeats, whereas Tfx1 serves to maintain these elevated levels. This reveals a novel mechanism for the regulation of telomeric transcripts. We extend this to demonstrate that human Translin and Trax also control telomere-associated transcript levels in human cells in a telomere-specific fashion.

Comprehensive nucleosome mapping of the human genome in cancer progression.

Altered chromatin structure is a hallmark of cancer, and inappropriate regulation of chromatin structure may represent the origin of transformation. Important studies have mapped human nucleosome distributions genome wide, but the role of chromatin structure in cancer progression has not been addressed. We developed a MNase-Transcription Start Site Sequence Capture method (mTSS-seq) to map the nucleosome distribution at human transcription start sites genome-wide in primary human lung and colon adenocarcinoma tissue. Here, we confirm that nucleosome redistribution is an early, widespread event in lung (LAC) and colon (CRC) adenocarcinoma. These altered nucleosome architectures are consistent between LAC and CRC patient samples indicating that they may serve as important early adenocarcinoma markers. We demonstrate that the nucleosome alterations are driven by the underlying DNA sequence and potentiate transcription factor binding. We conclude that DNA-directed nucleosome redistributions are widespread early in cancer progression. We have proposed an entirely new hierarchical model for chromatin-mediated genome regulation.

OncoBinder facilitates interpretation of proteomic interaction data by capturing coactivation pairs in cancer.

High-throughput methods such as co-immunoprecipitationmass spectrometry (coIP-MS) and yeast 2 hybridization (Y2H) have suggested a broad range of unannotated protein-protein interactions (PPIs), and interpretation of these PPIs remains a challenging task. The advancements in cancer genomic researches allow for the inference of "coactivation pairs" in cancer, which may facilitate the identification of PPIs involved in cancer. Here we present OncoBinder as a tool for the assessment of proteomic interaction data based on the functional synergy of oncoproteins in cancer. This decision tree-based method combines gene mutation, copy number and mRNA expression information to infer the functional status of protein-coding genes. We applied OncoBinder to evaluate the potential binders of EGFR and ERK2 proteins based on the gastric cancer dataset of The Cancer Genome Atlas (TCGA). As a result, OncoBinder identified high confidence interactions (annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) or validated by low-throughput assays) more efficiently than co-expression based method. Taken together, our results suggest that evaluation of gene functional synergy in cancer may facilitate the interpretation of proteomic interaction data. The OncoBinder toolbox for Matlab is freely accessible online.

Hierarchical regulation of the genome: global changes in nucleosome organization potentiate genome response.

Nucleosome occupancy is critically important in regulating access to the eukaryotic genome. Few studies in human cells have measured genome-wide nucleosome distributions at high temporal resolution during a response to a common stimulus. We measured nucleosome distributions at high temporal resolution following Kaposi's-sarcoma-associated herpesvirus (KSHV) reactivation using our newly developed mTSS-seq technology, which maps nucleosome distribution at the transcription start sites (TSS) of all human genes. Nucleosomes underwent widespread changes in organization 24 hours after KSHV reactivation and returned to their basal nucleosomal architecture 48 hours after KSHV reactivation. The widespread changes consisted of an indiscriminate remodeling event resulting in the loss of nucleosome rotational phasing signals. Additionally, one in six TSSs in the human genome possessed nucleosomes that are translationally remodeled. 72% of the loci with translationally remodeled nucleosomes have nucleosomes that moved to positions encoded by the underlying DNA sequence. Finally we demonstrated that these widespread alterations in nucleosomal architecture potentiated regulatory factor binding. These descriptions of nucleosomal architecture changes provide a new framework for understanding the role of chromatin in the genomic response, and have allowed us to propose a hierarchical model for chromatin-based regulation of genome response.

Chromatin determinants of the inner-centromere rely on replication factors with functions that impart cohesion.

Replication fork-associated factors promote genome integrity and protect against cancer. Mutations in the DDX11 helicase and the ESCO2 acetyltransferase also cause related developmental disorders classified as cohesinopathies. Here we generated vertebrate model cell lines of these disorders and cohesinopathies-related genes. We found that vertebrate DDX11 and Tim-Tipin are individually needed to compensate for ESCO2 loss in chromosome segregation, with DDX11 also playing complementary roles with ESCO2 in centromeric cohesion. Our study reveals that overt centromeric cohesion loss does not necessarily precede chromosome missegregation, while both these problems correlate with, and possibly originate from, inner-centromere defects involving reduced phosphorylation of histone H3T3 (pH3T3) in the region. Interestingly, the mitotic pH3T3 mark was defective in all analyzed replication-related mutants with functions in cohesion. The results pinpoint mitotic pH3T3 as a postreplicative chromatin mark that is sensitive to replication stress and conducts with different kinetics to robust centromeric cohesion and correct chromosome segregation.

Mcl-1 dynamics influence mitotic slippage and death in mitosis.

Microtubule-binding drugs such as taxol are frontline treatments for a variety of cancers but exactly how they yield patient benefit is unclear. In cell culture, inhibiting microtubule dynamics prevents spindle assembly, leading to mitotic arrest followed by either apoptosis in mitosis or slippage, whereby a cell returns to interphase without dividing. Myeloid cell leukaemia-1 (Mcl-1), a pro-survival member of the Bcl-2 family central to the intrinsic apoptosis pathway, is degraded during a prolonged mitotic arrest and may therefore act as a mitotic death timer. Consistently, we show that blocking proteasome-mediated degradation inhibits taxol-induced mitotic apoptosis in a Mcl-1-dependent manner. However, this degradation does not require the activity of either APC/C-Cdc20, FBW7 or MULE, three separate E3 ubiquitin ligases implicated in targeting Mcl-1 for degradation. This therefore challenges the notion that Mcl-1 undergoes regulated degradation during mitosis. We also show that Mcl-1 is continuously synthesized during mitosis and that blocking protein synthesis accelerates taxol induced death-in-mitosis. Modulating Mcl-1 levels also influences slippage; overexpressing Mcl-1 extends the time from mitotic entry to mitotic exit in the presence of taxol, while inhibiting Mcl-1 accelerates it. We suggest that Mcl-1 competes with Cyclin B1 for binding to components of the proteolysis machinery, thereby slowing down the slow degradation of Cyclin B1 responsible for slippage. Thus, modulating Mcl-1 dynamics influences both death-in-mitosis and slippage. However, because mitotic degradation of Mcl-1 appears not to be under the control of an E3 ligase, we suggest that the notion of network crosstalk is used with caution.

Polo-like kinase 1 mediates BRCA1 phosphorylation and recruitment at DNA double-strand breaks.

Accurate repair of DNA double-strand breaks (DSB) caused during DNA replication and by exogenous stresses is critical for the maintenance of genomic integrity. There is growing evidence that the Polo-like kinase 1 (Plk1) that plays a number of pivotal roles in cell proliferation can directly participate in regulation of DSB repair. In this study, we show that Plk1 regulates BRCA1, a key mediator protein required to efficiently repair DSB through homologous recombination (HR). Following induction of DSB, BRCA1 concentrates in distinctive large nuclear foci at damage sites where multiple DNA repair factors accumulate. First, we found that inhibition of Plk1 shortly before DNA damage sensitizes cells to ionizing radiation and reduces DSB repair by HR. Second, we provide evidence that BRCA1 foci formation induced by DSB is reduced when Plk1 is inhibited or depleted. Third, we identified BRCA1 as a novel Plk1 substrate and determined that Ser1164 is the major phosphorylation site for Plk1 in vitro. In cells, mutation of Plk1 sites on BRCA1 significantly delays BRCA1 foci formation following DSB, recapitulating the phenotype observed upon Plk1 inhibition. Our data then assign a key function to Plk1 in BRCA1 foci formation at DSB, emphasizing Plk1 importance in the HR repair of human cells.

Novel Mad2-targeting miR-493-3p controls mitotic fidelity and cancer cells' sensitivity to paclitaxel.

The molecular pathways that contribute to the proliferation and drug response of cancer cells are highly complex and currently insufficiently characterized. We have identified a previously unknown microRNA-based mechanism that provides cancer cells means to stimulate tumorigenesis via increased genomic instability and, at the same time, evade the action of clinically utilized microtubule drugs. We demonstrate miR-493-3p to be a novel negative regulator of mitotic arrest deficient-2 (MAD2), an essential component of the spindle assembly checkpoint that monitors the fidelity of chromosome segregation. The microRNA targets the 3' UTR of Mad2 mRNA thereby preventing translation of the Mad2 protein. In cancer cells, overexpression of miR-493-3p induced a premature mitotic exit that led to increased frequency of aneuploidy and cellular senescence in the progeny cells. Importantly, excess of the miR-493-3p conferred resistance of cancer cells to microtubule drugs. In human neoplasms, miR-493-3p and Mad2 expression alterations correlated with advanced ovarian cancer forms and high miR-493-3p levels were associated with reduced survival of ovarian and breast cancer patients with aggressive tumors, especially in the paclitaxel therapy arm. Our results suggest that intratumoral profiling of miR-493-3p and Mad2 levels can have diagnostic value in predicting the efficacy of taxane chemotherapy.

DNA demethylation caused by 5-Aza-2'-deoxycytidine induces mitotic alterations and aneuploidy.

Aneuploidy, the unbalanced number of chromosomes in a cell, is considered a prevalent form of genetic instability and is largely acknowledged as a condition implicated in tumorigenesis. Epigenetic alterations like DNA hypomethylation have been correlated with cancer initiation/progression. Furthermore, a growing body of evidence suggests the involvement of epigenome-wide disruption as a cause of global DNA hypomethylation in aneuploidy generation.Here, we report that the DNA hypomethylating drug 5-aza-2'-deoxycytidine (DAC), affects the correct ploidy of nearly diploid HCT-116 human cells by altering the methylation pattern of the chromosomes. Specifically, we show that a DAC-induced reduction of 5-Methyl Cytosine at the pericentromeric region of chromosomes correlates with aneuploidy and mitotic defects.Our results suggest that DNA hypomethylation leads to aneuploidy by altering the DNA methylation landscape at the centromere that is necessary to ensure proper chromosomes segregation by recruiting the proteins necessary to build up a functional kinetochore.

FBW7 regulates DNA interstrand cross-link repair by modulating FAAP20 degradation.

Mutations that deregulate protein degradation lead to human malignancies. The SCF ubiquitin E3 ligase complex degrades key oncogenic regulators, thereby limiting their oncogenic potential. FBW7 is a substrate recognition subunit of SCFFBW7 and is among the most commonly mutated ubiquitin-proteasome system proteins in cancer. FBW7-mutated cancer cells display increased genome instability, but the molecular mechanism by which FBW7 preserves genome integrity remains elusive. Here, we demonstrate that SCFFBW7 regulates the stability of FAAP20, a critical component of the Fanconi anemia (FA) DNA interstrand cross-link (ICL) repair pathway. Phosphorylation of the FAAP20 degron motif by GSK3ß provides a platform for recognition and polyubiquitination of FAAP20 by FBW7, and its subsequent degradation by the proteasome. Accordingly, enhanced GSK3ß-FBW7 signaling disrupts the FA pathway. In cells expressing non-phosphorylatable FAAP20 mutant, the turnover of its binding partner, FANCA, is deregulated in the chromatin during DNA ICL repair, and the FA pathway is compromised. We propose that FAAP20 degradation, which is prompted by its phosphorylation, controls the dynamics of the FA core complex required for completing DNA ICL repair. Together, this study provides insights into how FBW7-mediated proteolysis regulates genome stability and how its deregulation is associated with tumorigenesis.

Integration of microarray analysis into the clinical diagnosis of hematological malignancies: How much can we improve cytogenetic testing?

PURPOSE: To evaluate the clinical utility, diagnostic yield and rationale of integrating microarray analysis in the clinical diagnosis of hematological malignancies in comparison with classical chromosome karyotyping/fluorescence in situ hybridization (FISH). METHODS: G-banded chromosome analysis, FISH and microarray studies using customized CGH and CGH+SNP designs were performed on 27 samples from patients with hematological malignancies. A comprehensive comparison of the results obtained by three methods was conducted to evaluate benefits and limitations of these techniques for clinical diagnosis. RESULTS: Overall, 89.7% of chromosomal abnormalities identified by karyotyping/FISH studies were also detectable by microarray. Among 183 acquired copy number alterations (CNAs) identified by microarray, 94 were additional findings revealed in 14 cases (52%), and at least 30% of CNAs were in genomic regions of diagnostic/prognostic significance. Approximately 30% of novel alterations detected by microarray were >20 Mb in size. Balanced abnormalities were not detected by microarray; however, of the 19 apparently "balanced" rearrangements, 55% (6/11) of recurrent and 13% (1/8) of non-recurrent translocations had alterations at the breakpoints discovered by microarray. CONCLUSION: Microarray technology enables accurate, cost-effective and time-efficient whole-genome analysis at a resolution significantly higher than that of conventional karyotyping and FISH. Array-CGH showed advantage in identification of cryptic imbalances and detection of clonal aberrations in population of non-dividing cancer cells and samples with poor chromosome morphology. The integration of microarray analysis into the cytogenetic diagnosis of hematologic malignancies has the potential to improve patient management by providing clinicians with additional disease specific and potentially clinically actionable genomic alterations.

Proteasome inhibition mediates p53 reactivation and anti-cancer activity of 6-gingerol in cervical cancer cells.

Human papilloma virus (HPV) expressing E6 and E7 oncoproteins, is known to inactivate the tumor suppressor p53 through proteasomal degradation in cervical cancers. Therefore, use of small molecules for inhibition of proteasome function and induction of p53 reactivation is a promising strategy for induction of apoptosis in cervical cancer cells. The polyphenolic alkanone, 6-Gingerol (6G), present in the pungent extracts of ginger (Zingiber officinale Roscoe) has shown potent anti-tumorigenic and pro-apoptotic activities against a variety of cancers. In this study we explored the molecular mechanism of action of 6G in human cervical cancer cells in vitro and in vivo. 6G potently inhibited proliferation of the HPV positive cervical cancer cells. 6G was found to: (i) inhibit the chymotrypsin activity of proteasomes, (ii) induce reactivation of p53, (iii) increase levels of p21, (iv) induce DNA damage and G2/M cell cycle arrest, (v) alter expression levels of p53-associated apoptotic markers like, cleaved caspase-3 and PARP, and (vi) potentiate the cytotoxicity of cisplatin. 6G treatment induced significant reduction of tumor volume, tumor weight, proteasome inhibition and p53 accumulation in HeLa xenograft tumor cells in vivo. The 6G treatment was devoid of toxic effects as it did not affect body weights, hematological and osteogenic parameters. Taken together, our data underscores the therapeutic and chemosensitizing effects of 6G in the management and treatment of cervical cancer.

A novel retroviral mutagenesis screen identifies prognostic genes in RUNX1 mediated myeloid leukemogenesis.

Using a novel retroviral shuttle vector approach we identified genes that collaborate with a patient derived RUNX1 (AML1) mutant. RUNX1 mutations occurs in 40% of myelodysplastic syndromes (MDS). MDS are a group of hematopoietic stem cell disorders that are characterized by dysplasia that often progress to acute myeloid leukemia (AML). Our goal was to identify genes dysregulated by vector-mediated genotoxicity that may collaborate with the RUNX1 mutant (D171N). D171N expressing cells have a survival and engraftment disadvantage and require additional genetic lesions to survive and persist. By dysregulating genes near the integrated vector provirus, the shuttle vector can promote transformation of D171N cells and tag the nearby genes that collaborate with D171N. In our approach, a gammaretroviral shuttle vector that expresses D171N is used to transduce CD105+, Sca-1+ mouse bone marrow. Mutagenized cells are expanded in liquid culture and vector integration sites from surviving cells are then identified using a retroviral shuttle vector approach. We repeatedly recovered integrated vector proviruses near genes (Itpkb, Ccdc12, and Nbeal2). To assess the prognostic significance of the genes identified we examined differential expression, overall survival, and relapse free survival of AML patients with alteration in the genes identified using The Cancer Genome Atlas (TCGA) AML data set. We found that ITPKB functions as an independent factor for poor prognoses and RUNX1 mutations in conjunction with ITPKB, CCDC12, and NBEAL2 have prognostic potential in AML.

Genome-wide array-CGH analysis reveals YRF1 gene copy number variation that modulates genetic stability in distillery yeasts.

Industrial yeasts, economically important microorganisms, are widely used in diverse biotechnological processes including brewing, winemaking and distilling. In contrast to a well-established genome of brewer's and wine yeast strains, the comprehensive evaluation of genomic features of distillery strains is lacking. In the present study, twenty two distillery yeast strains were subjected to electrophoretic karyotyping and array-based comparative genomic hybridization (array-CGH). The strains analyzed were assigned to the Saccharomyces sensu stricto complex and grouped into four species categories: S. bayanus, S. paradoxus, S. cerevisiae and S. kudriavzevii. The genomic diversity was mainly revealed within subtelomeric regions and the losses and/or gains of fragments of chromosomes I, III, VI and IX were the most frequently observed. Statistically significant differences in the gene copy number were documented in six functional gene categories: 1) telomere maintenance via recombination, DNA helicase activity or DNA binding, 2) maltose metabolism process, glucose transmembrane transporter activity; 3) asparagine catabolism, cellular response to nitrogen starvation, localized in cell wall-bounded periplasmic space, 4) siderophore transport, 5) response to copper ion, cadmium ion binding and 6) L-iditol 2- dehydrogenase activity. The losses of YRF1 genes (Y' element ATP-dependent helicase) were accompanied by decreased level of Y' sequences and an increase in DNA double and single strand breaks, and oxidative DNA damage in the S. paradoxus group compared to the S. bayanus group. We postulate that naturally occurring diversity in the YRF1 gene copy number may promote genetic stability in the S. bayanus group of distillery yeast strains.

Cdk5 and Foxc2--a new relationship in the lymphatic vasculature.

Lymphatic vessel dysfunction is associated with various pathologic conditions, including immunologic disorders, lymphedema, as well as tumor dissemination. Yet, the knowledge about the regulation of lymphatic vessel development is still limited. Our study elucidates cyclin dependent kinase 5 (Cdk5) as an essential player in the development of lymphatic vessels. Deletion of Cdk5 in the mouse endothelium results in severe lymphedema formation and embryonic lethality. On the mechanistic level, we show that Cdk5 phosphorylates the forkhead transcription factor Foxc2 which regulates Foxc2-dependent transcription. In summary, our study elucidates the Cdk5-Foxc2 interaction as a critical regulator of lymphatic vessel development.