DNA and histone modifications combine into characteristic patterns that demarcate functional regions of the genome^1,2. While many ‘readers’ of individual modifications have been described^3–5, how chromatin states comprising composite modification signatures, histone variants and internucleosomal linker DNA are interpreted is a major open question. Here we use a multidimensional proteomics strategy to systematically examine the interaction of around 2,000 nuclear proteins with over 80 modified dinucleosomes representing promoter, enhancer and heterochromatin states. By deconvoluting complex nucleosome-binding profiles into networks of co-regulated proteins and distinct nucleosomal features driving protein recruitment or exclusion, we show comprehensively how chromatin states are decoded by chromatin readers. We find highly distinctive binding responses to different features, many factors that recognize multiple features, and that nucleosomal modifications and linker DNA operate largely independently in regulating protein binding to chromatin. Our online resource, the Modification Atlas of Regulation by Chromatin States (MARCS), provides in-depth analysis tools to engage with our results and advance the discovery of fundamental principles of genome regulation by chromatin states.

Avian cryptochrome 4 (Cry4) protein is a putative magnetosensitive molecule facilitating precise long-distance navigation in migratory birds. Two splice variants of Cry4 have been reported in the European robin ( Erithacus rubecula ), namely Er Cry4a and Er Cry4b. Er Cry4a protein is known to generate magnetically sensitive radical pairs for magnetoreception through electron transfer between the flavin adenine dinucleotide (FAD) cofactor and tryptophan residues within the protein. However, little is known about the Er Cry4b isoform. We therefore characterize the properties of Er Cry4b to determine whether it fulfils the prerequisites to be a radical-pair magnetic sensor molecule. Our results show that Er Cry4b protein does not bind FAD in vitro . Computational simulations reveal that the lack of FAD binding in Er Cry4b is probably due to protein structural dynamics. Furthermore, immunoprecipitation assays coupled with mass spectrometry suggest that Er Cry4b protein is undetectable in the robin retina, cerebellum, and liver. Meanwhile, transcript analyses show that ErCRY4b mRNA abundance is 10 times lower than that of ErCRY4a in the retina. In conclusion, Er Cry4b does not fulfil the prerequisites to be a radical pair-based magnetic sensing molecule due to the lack of FAD binding, and it might not even be expressed as a functional protein in the European robin.

Combined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy (ET) improves outcomes in advanced estrogen receptor–positive (ER ⁺ ) breast cancer, but emergence of resistance to this combination underscores the pressing need for alternative therapeutic strategies. A promising approach involves adding an inhibitor of the PI3K/AKT/mTOR pathway to the standard combined CDK4/6i and ET, but selecting the most effective inhibitors and their optimal combinations has proven to be challenging. Here, we compared the efficacy of various triple combinations using single- or dual-point PI3K/AKT/mTOR pathway inhibitors in breast cancer cell lines, cell line xenografts, patient-derived xenografts, and organoids resistant to CDK4/6i and ET and exhibiting PIK3CA , PTEN , or AKT1 mutations. PIK3CA -mutant, PTEN –wild type, CDK4/6i-resistant, and ET-resistant models required the addition of the dual PI3K/mTOR inhibitor gedatolisib to effectively impede tumor growth by blocking the HIF-1α pathway through both mTORC1 inhibition and PI3K/AKT-mediated modulation of GSK3α/β activity. Conversely, PIK3CA –wild type, PTEN -null cells benefited from triple combinations incorporating either the AKT inhibitor capivasertib or the dual mTORC1/2 inhibitor sapanisertib to block tumor growth. In addition, gedatolisib reduced viability of PIK3CA - or AKT1 -mutant and PTEN –wild type CDK4/6i-resistant patient-derived organoids compared with the α-specific PI3K inhibitor alpelisib. Our data support the higher efficacy of gedatolisib over alpelisib in ER ⁺ breast tumors harboring alterations of the PI3K/AKT/mTOR pathway including PIK3CA or AKT1 mutations.

Eukaryotic genome replication is surveyed by the S-phase checkpoint, which coordinates sequential origin activation to prevent the exhaustion of poorly defined, rate-limiting replisome components ^1–3 . Here we show that excessive origin firing saturates chromatin-bound proliferating cell nuclear antigen (PCNA)—a sliding clamp for DNA polymerase processivity and Okazaki fragment processing ⁴ —thereby restricting further PCNA loading and lagging-strand synthesis when checkpoint control is lost. PCNA-associated factor 15 (PAF15) emerges as a dosage-sensitive regulator of this process ^5–9 . During unperturbed S phase, the entire soluble PAF15 pool binds to chromatin, leaving no reserve to stabilize PCNA under conditions of excessive origin activation. PAF15 binds to PCNA specifically on the lagging strand through a high-affinity PIP motif and occupies the DNA-encircling channel, protecting the clamp and associated enzymes from premature unloading by the ATAD5–RFC complex. Conversely, overexpression of PAF15 or forced redistribution to the leading strand disrupts replisome progression and induces cell death. These detrimental effects are mitigated by Timeless–Claspin, which blocks PAF15–PCNA binding on the leading strand. E2F4-mediated repression fine-tunes PAF15 expression to ensure optimal dosage and strand specificity. These findings reveal a previously unrecognized replisome constraint: when PAF15–PCNA assemblies are exhausted, the S-phase checkpoint globally restricts origin activation, linking a strand-specific rate-limiting mechanism to global replication dynamics.

Triple-negative breast cancer (TNBC) is a particularly aggressive subtype of breast cancer with high metastatic potential, limited treatment options, and low patient survival rates. By combining functional proteomic and genomic approaches, we identified an oncogenic transcriptional network in mesenchymal and invasive TNBC involving the glucocorticoid receptor, GATA6, MYC, and AP-1 transcription factors. Although these transcription factors bound extensively to shared enhancers, they utilized different enhancer repertoires from this shared enhancer pool to drive distinct downstream oncogenic pathways. The SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex was a common gatekeeper of chromatin access for these transcription factors, and SWI/SNF inhibition decommissioned the distinct enhancer networks and protumorigenic gene programs they control. Consistently, SWI/SNF inhibition repressed TNBC proliferation, glucocorticoid-induced chemoresistance, and invasion in vitro and suppressed tumor growth and metastasis in vivo. Thus, these findings indicate that SWI/SNF-mediated chromatin remodeling drives TNBC progression, positioning SWI/SNF as a promising therapeutic target in this disease. Significance: Inhibition of SWI/SNF-mediated chromatin remodeling represses multiple cancer hallmarks in triple-negative breast cancer by blocking chromatin access for diverse transcription factors at shared enhancers, demonstrating the therapeutic potential of targeting DNA access.