Cdc48 and its co-factor Ufd1 extract CENP-A from centromeric chromatin and can induce chromosome elimination in the fission yeast Schizosaccharomyces pombe.

CENP-A determines the identity of the centromere. Because the position and size of the centromere and its number per chromosome must be maintained, the distribution of CENP-A is strictly regulated. In this study, we have aimed to understand mechanisms to regulate the distribution of CENP-A (Cnp1SP) in fission yeast. A mutant of the ufd1+ gene (ufd1-73) encoding a cofactor of Cdc48 ATPase is sensitive to Cnp1 expressed at a high level and allows mislocalization of Cnp1. The level of Cnp1 in centromeric chromatin is increased in the ufd1-73 mutant even when Cnp1 is expressed at a normal level. A preexisting mutant of the cdc48+ gene (cdc48-353) phenocopies the ufd1-73 mutant. We have also shown that Cdc48 and Ufd1 proteins interact physically with centromeric chromatin. Finally, Cdc48 ATPase with Ufd1 artificially recruited to the centromere of a mini-chromosome (Ch16) induce a loss of Cnp1 from Ch16, leading to an increased rate of chromosome loss. It appears that Cdc48 ATPase, together with its cofactor Ufd1 remove excess Cnp1 from chromatin, likely in a direct manner. This mechanism may play a role in centromere disassembly, a process to eliminate Cnp1 to inactivate the kinetochore function during development, differentiation, and stress response.

CENPA promotes glutamine metabolism and tumor progression by up-regulating SLC38A1 in endometrial cancer.

Glutamine addiction is a significant hallmark of metabolic reprogramming in tumors and is crucial to the progression of cancer. Nevertheless, the regulatory mechanisms of glutamine metabolism in endometrial cancer (EC) remains elusive. In this research, we found that elevated expression of CENPA and solute carrier family 38 member 1 (SLC38A1) were firmly associated with worse clinical stage and unfavorable outcomes in EC patients. In addition, ectopic overexpression or silencing of CENPA could either enhance or diminish glutamine metabolism and tumor progression in EC. Mechanistically, CENPA directly regulated the transcriptional activity of the target gene, SLC38A1, leading to enhanced glutamine uptake and metabolism, thereby promoting EC progression. Notably, a prognostic model utilizing the expression levels of CENPA and SLC38A1 genes independently emerged as a prognostic factor for EC. More importantly, CENPA and SLC38A1 were significantly elevated and positively correlated, as well as indicative of poor prognosis in multiple cancers. In brief, our study confirmed that CENPA is a critical transcription factor involved in glutamine metabolism and tumor progression through modulating SLC38A1. This revelation suggests that targeting CENPA could be an appealing therapeutic approach to address pan-cancer glutamine addiction.

Artificial tethering of constitutive centromere-associated network proteins induces CENP-A deposition without Knl2 in DT40 cells.

The kinetochore is an essential structure for chromosome segregation. Although the kinetochore is usually formed on a centromere locus, it can be artificially formed at a non-centromere locus by protein tethering. An artificial kinetochore can be formed by tethering of CENP-C or CENP-I, members of the constitutive centromere-associated network (CCAN). However, how CENP-C or CENP-I recruit the centromere-specific histone CENP-A to form an artificial kinetochore remains unclear. In this study, we analyzed this issue using the tethering assay combined with an auxin-inducible degron (AID)-based knockout method in chicken DT40 cells. We found that tethering of CENP-C or CENP-I induced CENP-A incorporation at the non-centromeric locus in the absence of Knl2 (or MIS18BP1), a component of the Mis18 complex, and that Knl2 tethering recruited CENP-A in the absence of CENP-C. We also showed that CENP-C coimmunoprecipitated with HJURP, independently of Knl2. Considering these results, we propose that CENP-C recruits CENP-A by HJURP binding to form an artificial kinetochore. Our results suggest that CENP-C or CENP-I exert CENP-A recruitment activity, independently of Knl2, for artificial kinetochore formation in chicken DT40 cells. This gives us a new insight into mechanisms for CENP-A incorporation.

Fluorescence complementation-based FRET imaging reveals centromere assembly dynamics.

Visualization of specific molecules and their assembly in real time and space is essential to delineate how cellular dynamics and signaling circuit are orchestrated during cell division cycle. Our recent studies reveal structural insights into human centromere-kinetochore core CCAN complex. Here we introduce a method for optically imaging trimeric and tetrameric protein interactions at nanometer spatial resolution in live cells using fluorescence complementation-based Förster resonance energy transfer (FC-FRET). Complementary fluorescent protein molecules were first used to visualize dimerization followed by FRET measurements. Using FC-FRET, we visualized centromere CENP-SXTW tetramer assembly dynamics in live cells, and dimeric interactions between CENP-TW dimer and kinetochore protein Spc24/25 dimer in dividing cells. We further delineated the interactions of monomeric CENP-T with Spc24/25 dimer in dividing cells. Surprisingly, our analyses revealed critical role of CDK1 kinase activity in the initial recruitment of Spc24/25 by CENP-T. However, interactions between CENP-T and Spc24/25 during chromosome segregation is independent of CDK1. Thus, FC-FRET provides a unique approach to delineate spatiotemporal dynamics of trimerized and tetramerized proteins at nanometer scale and establishes a platform to report the precise regulation of multimeric protein interactions in space and time in live cells.

CENPA knockdown restrains cell progression and tumor growth in breast cancer by reducing PLA2R1 promoter methylation and modulating PLA2R1/HHEX axis.

BACKGROUND: Breast cancer is a lethal malignancy affecting females worldwide. It has been reported that upregulated centromere protein A (CENPA) expression might indicate unfortunate prognosis and can function as a prognostic biomarker in breast cancer. This study aimed to investigate the accurate roles and downstream mechanisms of CENPA in breast cancer progression. METHODS: CENPA protein levels in breast cancer tissues and cell lines were analyzed by Western blot and immunohistochemistry assays. We used gain/loss-of-function experiments to determine the potential effects of CENPA and phospholipase A2 receptor (PLA2R1) on breast cancer cell proliferation, migration, and apoptosis. Co-IP assay was employed to validate the possible interaction between CENPA and DNA methyltransferase 1 (DNMT1), as well as PLA2R1 and hematopoietically expressed homeobox (HHEX). PLA2R1 promoter methylation was determined using methylation-specific PCR assay. The biological capabilities of CENPA/PLA2R1/HHEX axis in breast cancer cells was determined by rescue experiments. In addition, CENPA-silenced MCF-7 cells were injected into mice, followed by measurement of tumor growth. RESULTS: CENPA level was prominently elevated in breast cancer tissues and cell lines. Interestingly, CENPA knockdown and PLA2R1 overexpression both restrained breast cancer cell proliferation and migration, and enhanced apoptosis. On the contrary, CENPA overexpression displayed the opposite results. Moreover, CENPA reduced PLA2R1 expression through promoting DNMT1-mediated PLA2R1 promoter methylation. PLA2R1 overexpression could effectively abrogate CENPA overexpression-mediated augment of breast cancer cell progression. Furthermore, PLA2R1 interacted with HHEX and promoted HHEX expression. PLA2R1 knockdown increased the rate of breast cancer cell proliferation and migration but restrained apoptosis, which was abrogated by HHEX overexpression. In addition, CENPA silencing suppressed tumor growth in vivo. CONCLUSION: CENPA knockdown restrained breast cancer cell proliferation and migration and attenuated tumor growth in vivo through reducing PLA2R1 promoter methylation and increasing PLA2R1 and HHEX expression. We may provide a promising prognostic biomarker and novel therapeutic target for breast cancer.

HJURP is recruited to double-strand break sites and facilitates DNA repair by promoting chromatin reorganization.

HJURP is overexpressed in several cancer types and strongly correlates with patient survival. However, the mechanistic basis underlying the association of HJURP with cancer aggressiveness is not well understood. HJURP promotes the loading of the histone H3 variant, CENP-A, at the centromeric chromatin, epigenetically defining the centromeres and supporting proper chromosome segregation. In addition, HJURP is associated with DNA repair but its function in this process is still scarcely explored. Here, we demonstrate that HJURP is recruited to DSBs through a mechanism requiring chromatin PARylation and promotes epigenetic alterations that favor the execution of DNA repair. Incorporation of HJURP at DSBs promotes turnover of H3K9me3 and HP1, facilitating DNA damage signaling and DSB repair. Moreover, HJURP overexpression in glioma cell lines also affected global structure of heterochromatin independently of DNA damage induction, promoting genome-wide reorganization and assisting DNA damage response. HJURP overexpression therefore extensively alters DNA damage signaling and DSB repair, and also increases radioresistance of glioma cells. Importantly, HJURP expression levels in tumors are also associated with poor response of patients to radiation. Thus, our results enlarge the understanding of HJURP involvement in DNA repair and highlight it as a promising target for the development of adjuvant therapies that sensitize tumor cells to irradiation.

Interaction of histone H4 with Cse4 facilitates conformational changes in Cse4 for its sumoylation and mislocalization.

Mislocalization of overexpressed CENP-A (Cse4 in budding yeast, Cnp1 in fission yeast, CID in flies) contributes to chromosomal instability (CIN) in yeasts, flies, and human cells. Mislocalization of CENP-A is observed in many cancers and this correlates with poor prognosis. Structural mechanisms that contribute to mislocalization of CENP-A are poorly defined. Here, we show that interaction of histone H4 with Cse4 facilitates an in vivo conformational change in Cse4 promoting its mislocalization in budding yeast. We determined that Cse4 Y193A mutant exhibits reduced sumoylation, mislocalization, interaction with histone H4, and lethality in psh1Δ and cdc48-3 strains; all these phenotypes are suppressed by increased gene dosage of histone H4. We developed a new in vivo approach, antibody accessibility (AA) assay, to examine the conformation of Cse4. AA assay showed that wild-type Cse4 with histone H4 is in an 'open' state, while Cse4 Y193A predominantly exhibits a 'closed' state. Increased gene dosage of histone H4 contributes to a shift of Cse4 Y193A to an 'open' state with enhanced sumoylation and mislocalization. We provide molecular insights into how Cse4-H4 interaction changes the conformational state of Cse4 in vivo. These studies advance our understanding for mechanisms that promote mislocalization of CENP-A in human cancers.

The cysteine-rich domain in CENP-A chaperone Scm3HJURP ensures centromere targeting and kinetochore integrity.

Centromeric chromatin plays a crucial role in kinetochore assembly and chromosome segregation. Centromeres are specified through the loading of the histone H3 variant CENP-A by the conserved chaperone Scm3/HJURP. The N-terminus of Scm3/HJURP interacts with CENP-A, while the C-terminus facilitates centromere localization by interacting with the Mis18 holocomplex via a small domain, called the Mis16-binding domain (Mis16-BD) in fission yeast. Fungal Scm3 proteins contain an additional conserved cysteine-rich domain (CYS) of unknown function. Here, we find that CYS binds zinc in vitro and is essential for the localization and function of fission yeast Scm3. Disrupting CYS by deletion or introduction of point mutations within its zinc-binding motif prevents Scm3 centromere localization and compromises kinetochore integrity. Interestingly, CYS alone can localize to the centromere, albeit weakly, but its targeting is greatly enhanced when combined with Mis16-BD. Expressing a truncated protein containing both Mis16-BD and CYS, but lacking the CENP-A binding domain, causes toxicity and is accompanied by considerable chromosome missegregation and kinetochore loss. These effects can be mitigated by mutating the CYS zinc-binding motif. Collectively, our findings establish the essential role of the cysteine-rich domain in fungal Scm3 proteins and provide valuable insights into the mechanism of Scm3 centromere targeting.

CENP-A and CENP-B collaborate to create an open centromeric chromatin state.

Centromeres are epigenetically defined via the presence of the histone H3 variant CENP-A. Contacting CENP-A nucleosomes, the constitutive centromere associated network (CCAN) and the kinetochore assemble, connecting the centromere to spindle microtubules during cell division. The DNA-binding centromeric protein CENP-B is involved in maintaining centromere stability and, together with CENP-A, shapes the centromeric chromatin state. The nanoscale organization of centromeric chromatin is not well understood. Here, we use single-molecule fluorescence and cryoelectron microscopy (cryoEM) to show that CENP-A incorporation establishes a dynamic and open chromatin state. The increased dynamics of CENP-A chromatin create an opening for CENP-B DNA access. In turn, bound CENP-B further opens the chromatin fiber structure and induces nucleosomal DNA unwrapping. Finally, removal of CENP-A increases CENP-B mobility in cells. Together, our studies show that the two centromere-specific proteins collaborate to reshape chromatin structure, enabling the binding of centromeric factors and establishing a centromeric chromatin state.

CENPA functions as a transcriptional regulator to promote hepatocellular carcinoma progression via cooperating with YY1.

The centromere proteins (CENPs), a critical mitosis-related protein complexes, are involved in the kinetochore assembly and chromosome segregation. In this study, we identified that CENPA was significantly up-regulated in HCC and highly expressed CENPA correlated with poor prognosis for HCC patients. Knockdown of CENPA inhibited HCC cell proliferation and tumor growth in vitro and in vivo. Mechanistically, CENPA transcriptionally activated and cooperated with YY1 to drive the expression of cyclin D1 (CCND1) and neuropilin 2 (NRP2). Moreover, we identified that CENPA can be lactylated at lysine 124 (K124). The lactylation of CENPA at K124 promotes CENPA activation, leading to enhanced expression of its target genes. In summary, CENPA function as a transcriptional regulator to promote HCC via cooperating with YY1. Targeting the CENPA-YY1-CCND1/NRP2 axis may provide candidate therapeutic targets for HCC.

Humanization reveals pervasive incompatibility of yeast and human kinetochore components.

Kinetochores assemble on centromeres to drive chromosome segregation in eukaryotic cells. Humans and budding yeast share most of the structural subunits of the kinetochore, whereas protein sequences have diverged considerably. The conserved centromeric histone H3 variant, CenH3 (CENP-A in humans and Cse4 in budding yeast), marks the site for kinetochore assembly in most species. A previous effort to complement Cse4 in yeast with human CENP-A was unsuccessful; however, co-complementation with the human core nucleosome was not attempted. Previously, our lab successfully humanized the core nucleosome in yeast; however, this severely affected cellular growth. We hypothesized that yeast Cse4 is incompatible with humanized nucleosomes and that the kinetochore represented a limiting factor for efficient histone humanization. Thus, we argued that including the human CENP-A or a Cse4-CENP-A chimera might improve histone humanization and facilitate kinetochore function in humanized yeast. The opposite was true: CENP-A expression reduced histone humanization efficiency, was toxic to yeast, and disrupted cell cycle progression and kinetochore function in wild-type (WT) cells. Suppressors of CENP-A toxicity included gene deletions of subunits of 3 conserved chromatin remodeling complexes, highlighting their role in CenH3 chromatin positioning. Finally, we attempted to complement the subunits of the NDC80 kinetochore complex, individually and in combination, without success, in contrast to a previous study indicating complementation by the human NDC80/HEC1 gene. Our results suggest that limited protein sequence similarity between yeast and human components in this very complex structure leads to failure of complementation.

Recognition of centromere-specific histone Cse4 by the inner kinetochore Okp1-Ame1 complex.

Successful mitosis depends on the timely establishment of correct chromosomal attachments to microtubules. The kinetochore, a modular multiprotein complex, mediates this connection by recognizing specialized chromatin containing a histone H3 variant called Cse4 in budding yeast and CENP-A in vertebrates. Structural features of the kinetochore that enable discrimination between Cse4/CENP-A and H3 have been identified in several species. How and when these contribute to centromere recognition and how they relate to the overall structure of the inner kinetochore are unsettled questions. More generally, this molecular recognition ensures that only one kinetochore is built on each chromatid and that this happens at the right place on the chromatin fiber. We have determined the crystal structure of a Cse4 peptide bound to the essential inner kinetochore Okp1-Ame1 heterodimer from budding yeast. The structure and related experiments show in detail an essential point of Cse4 contact and provide information about the arrangement of the inner kinetochore.

A dynamic population of prophase CENP-C is required for meiotic chromosome segregation.

The centromere is an epigenetic mark that is a loading site for the kinetochore during meiosis and mitosis. This mark is characterized by the H3 variant CENP-A, known as CID in Drosophila. In Drosophila, CENP-C is critical for maintaining CID at the centromeres and directly recruits outer kinetochore proteins after nuclear envelope break down. These two functions, however, happen at different times in the cell cycle. Furthermore, in Drosophila and many other metazoan oocytes, centromere maintenance and kinetochore assembly are separated by an extended prophase. We have investigated the dynamics of function of CENP-C during the extended meiotic prophase of Drosophila oocytes and found that maintaining high levels of CENP-C for metaphase I requires expression during prophase. In contrast, CID is relatively stable and does not need to be expressed during prophase to remain at high levels in metaphase I of meiosis. Expression of CID during prophase can even be deleterious, causing ectopic localization to non-centromeric chromatin, abnormal meiosis and sterility. CENP-C prophase loading is required for multiple meiotic functions. In early meiotic prophase, CENP-C loading is required for sister centromere cohesion and centromere clustering. In late meiotic prophase, CENP-C loading is required to recruit kinetochore proteins. CENP-C is one of the few proteins identified in which expression during prophase is required for meiotic chromosome segregation. An implication of these results is that the failure to maintain recruitment of CENP-C during the extended prophase in oocytes would result in chromosome segregation errors in oocytes.

Variation in the CENP-A sequence association landscape across diverse inbred mouse strains.

Centromeres are crucial for chromosome segregation, but their underlying sequences evolve rapidly, imposing strong selection for compensatory changes in centromere-associated kinetochore proteins to assure the stability of genome transmission. While this co-evolution is well documented between species, it remains unknown whether population-level centromere diversity leads to functional differences in kinetochore protein association. Mice (Mus musculus) exhibit remarkable variation in centromere size and sequence, but the amino acid sequence of the kinetochore protein CENP-A is conserved. Here, we apply k-mer-based analyses to CENP-A chromatin profiling data from diverse inbred mouse strains to investigate the interplay between centromere variation and kinetochore protein sequence association. We show that centromere sequence diversity is associated with strain-level differences in both CENP-A positioning and sequence preference along the mouse core centromere satellite. Our findings reveal intraspecies sequence-dependent differences in CENP-A/centromere association and open additional perspectives for understanding centromere-mediated variation in genome stability.

Direct recruitment of Mis18 to interphase spindle pole bodies promotes CENP-A chromatin assembly.

CENP-A chromatin specifies mammalian centromere identity, and its chaperone HJURP replenishes CENP-A when recruited by the Mis18 complex (Mis18C) via M18BP1/KNL2 to CENP-C at kinetochores during interphase. However, the Mis18C recruitment mechanism remains unresolved in species lacking M18BP1, such as fission yeast. Fission yeast centromeres cluster at G2 spindle pole bodies (SPBs) when CENP-ACnp1 is replenished and where Mis18C also localizes. We show that SPBs play an unexpected role in concentrating Mis18C near centromeres through the recruitment of Mis18 by direct binding to the major SPB linker of nucleoskeleton and cytoskeleton (LINC) component Sad1. Mis18C recruitment by Sad1 is important for CENP-ACnp1 chromatin establishment and acts in parallel with a CENP-C-mediated Mis18C recruitment pathway to maintain centromeric CENP-ACnp1 but operates independently of Sad1-mediated centromere clustering. SPBs therefore provide a non-chromosomal scaffold for both Mis18C recruitment and centromere clustering during G2. This centromere-independent Mis18-SPB recruitment provides a mechanism that governs de novo CENP-ACnp1 chromatin assembly by the proximity of appropriate sequences to SPBs and highlights how nuclear spatial organization influences centromere identity.

High expression of centromere protein A and its molecular mechanism and clinical significance in prostate cancer: A study based on data mining and immunohistochemistry.

The progression of prostate cancer (PCa) leads to poor prognosis. However, the molecular mechanism of PCa is still not completely clear. This study aimed to elucidate the important role of centromere protein A (CENPA) in PCa. Large numbers of bulk RNA sequencing (RNA-seq) data and in-house immunohistochemistry data were used in analysing the expression level of CENPA in PCa and metastatic PCa (MPCa). Single-cell RNA-seq data was used to explore the expression status of CENPA in different prostate subpopulations. Enrichment analysis was employed to detect the function of CENPA in PCa. Clinicopathological parameters analysis was utilised in analysing the clinical value of CENPA. The results showed that CENPA was upregulated in PCa (standardised mean difference [SMD] = 0.83, p = 0.001) and MPCa (SMD = 0.61, p = 0.029). CENPA was overexpressed in prostate cancer stem cells (CSCs) with androgen receptor (AR) negative compared to epithelial cells with AR positive. CENPA may influence the development of PCa through affecting cell cycle. Patients with nodal metastasis had higher expression level of CENPA. And patients with high CENPA expression had poor disease-free survival. Taken together, Overexpression of CENPA may influence the development of PCa by regulating cell cycle and promoting metastasis.

Misregulation of cell cycle-dependent methylation of budding yeast CENP-A contributes to chromosomal instability.

Centromere (CEN) identity is specified epigenetically by specialized nucleosomes containing evolutionarily conserved CEN-specific histone H3 variant CENP-A (Cse4 in Saccharomyces cerevisiae, CENP-A in humans), which is essential for faithful chromosome segregation. However, the epigenetic mechanisms that regulate Cse4 function have not been fully defined. In this study, we show that cell cycle-dependent methylation of Cse4-R37 regulates kinetochore function and high-fidelity chromosome segregation. We generated a custom antibody that specifically recognizes methylated Cse4-R37 and showed that methylation of Cse4 is cell cycle regulated with maximum levels of methylated Cse4-R37 and its enrichment at the CEN chromatin occur in the mitotic cells. Methyl-mimic cse4-R37F mutant exhibits synthetic lethality with kinetochore mutants, reduced levels of CEN-associated kinetochore proteins and chromosome instability (CIN), suggesting that mimicking the methylation of Cse4-R37 throughout the cell cycle is detrimental to faithful chromosome segregation. Our results showed that SPOUT methyltransferase Upa1 contributes to methylation of Cse4-R37 and overexpression of UPA1 leads to CIN phenotype. In summary, our studies have defined a role for cell cycle-regulated methylation of Cse4 in high-fidelity chromosome segregation and highlight an important role of epigenetic modifications such as methylation of kinetochore proteins in preventing CIN, an important hallmark of human cancers.

Direct observation of coordinated assembly of individual native centromeric nucleosomes.

Eukaryotic chromosome segregation requires the kinetochore, a megadalton-sized machine that forms on specialized centromeric chromatin containing CENP-A, a histone H3 variant. CENP-A deposition requires a chaperone protein HJURP that targets it to the centromere, but it has remained unclear whether HJURP has additional functions beyond CENP-A targeting and why high AT DNA content, which disfavors nucleosome assembly, is widely conserved at centromeres. To overcome the difficulties of studying nucleosome formation in vivo, we developed a microscopy assay that enables direct observation of de novo centromeric nucleosome recruitment and maintenance with single molecule resolution. Using this assay, we discover that CENP-A can arrive at centromeres without its dedicated centromere-specific chaperone HJURP, but stable incorporation depends on HJURP and additional DNA-binding proteins of the inner kinetochore. We also show that homopolymer AT runs in the yeast centromeres are essential for efficient CENP-A deposition. Together, our findings reveal requirements for stable nucleosome formation and provide a foundation for further studies of the assembly and dynamics of native kinetochore complexes.

Centromere/kinetochore is assembled through CENP-C oligomerization.

Kinetochore is an essential protein complex required for accurate chromosome segregation. The constitutive centromere-associated network (CCAN), a subcomplex of the kinetochore, associates with centromeric chromatin and provides a platform for the kinetochore assembly. The CCAN protein CENP-C is thought to be a central hub for the centromere/kinetochore organization. However, the role of CENP-C in CCAN assembly needs to be elucidated. Here, we demonstrate that both the CCAN-binding domain and the C-terminal region that includes the Cupin domain of CENP-C are necessary and sufficient for chicken CENP-C function. Structural and biochemical analyses reveal self-oligomerization of the Cupin domains of chicken and human CENP-C. We find that the CENP-C Cupin domain oligomerization is vital for CENP-C function, centromeric localization of CCAN, and centromeric chromatin organization. These results suggest that CENP-C facilitates the centromere/kinetochore assembly through its oligomerization.

MLL methyltransferases regulate H3K4 methylation to ensure CENP-A assembly at human centromeres.

The active state of centromeres is epigenetically defined by the presence of CENP-A interspersed with histone H3 nucleosomes. While the importance of dimethylation of H3K4 for centromeric transcription has been highlighted in various studies, the identity of the enzyme(s) depositing these marks on the centromere is still unknown. The MLL (KMT2) family plays a crucial role in RNA polymerase II (Pol II)-mediated gene regulation by methylating H3K4. Here, we report that MLL methyltransferases regulate transcription of human centromeres. CRISPR-mediated down-regulation of MLL causes loss of H3K4me2, resulting in an altered epigenetic chromatin state of the centromeres. Intriguingly, our results reveal that loss of MLL, but not SETD1A, increases co-transcriptional R-loop formation, and Pol II accumulation at the centromeres. Finally, we report that the presence of MLL and SETD1A is crucial for kinetochore maintenance. Altogether, our data reveal a novel molecular framework where both the H3K4 methylation mark and the methyltransferases regulate stability and identity of the centromere.