Biology subjects

Pihlajamaa, P.

Publications and source records attributed to Pihlajamaa, P..

6 recordsLinked to original sources

bioRxiv2026

Classical enhancers couple cis-regulatory logic with transcriptional condensates and 3D genome architecture

Deciphering the regulatory logic of enhancers remains a central question in understanding cell- and tissue-specific gene expression in multicellular organisms. This is particularly pertinent at multipartite enhancer clusters such as super-enhancers, where multiple enhancers contribute to the expression of a single gene. Gene expression has been studied largely through sequence-dependent recruitment of transcription factors (TF) and co-activators, whereas 3D chromatin structure has been attributed to architectural proteins such as cohesion and CTCF1-3. However, the contribution of DNA sequence encoded in enhancers to shaping higher-order genome organization remains poorly understood. Here we show that classical enhancers, embedded within multipartite super-enhancer structures, act as determinant regulatory elements that initiate the gene regulatory cascade by linking DNA sequence recognition to 3D chromatin architecture. Classical enhancers are more evolutionarily conserved and display stronger regulatory activity than facilitator elements, which lack intrinsic enhancer activity but potentiate classical enhancer function. We show that classical enhancers are selectively bound by specific TFs with strong intrinsically disordered regions, such as NFE2L2 in liver cancer cells, capable of driving transcriptional condensate formation through phase separation. NFE2L2 depletion reduced enhancer activity and induced widespread chromatin reorganization, characterized by increased cohesin and CTCF occupancy at super-enhancer boundaries and beyond. This "cohesin clogging" impaired DNA loop extrusion, led to formation of smaller topologically associated domains, and weakened enhancer-promoter contacts. These findings highlight that sequence-specific TFs have multifaceted roles beyond transcriptional control, establishing a direct mechanistic link between enhancer sequence, TF binding, condensate formation, and 3D genome organization, with the regulatory logic being encoded in the DNA sequence itself.

genomics↗

bioRxiv2024

Cancer cell type-specific derepression of transposable elements by inhibition of chromatin modifier enzymes

The combination of immunotherapy and epigenetic therapy is emerging as a promising approach for cancer therapy. Epigenetic therapy can induce derepression of transposable elements (TEs) that play a major role in activation of immune response against cancer cells. However, the molecular mechanism of TE regulation by distinct chromatin modifier enzymes (CME) and in the context of p53 is still elusive. Here, we used epigenetic drugs to inhibit distinct CMEs in p53 wild-type and p53-mutant colorectal and esophageal cancer cells. We show that distinct TEs subfamilies are derepressed by inhibition of different CMEs in a cell-type specific manner with loss of p53 resulting in stronger TE derepression. We show that KAP1, a known repressor of TEs, associates with stronger derepression of specific TE subfamilies such as LTR12C, indicating that KAP1 also has an activating role in TE regulation in cancer cells upon co-inhibition of DNMT and HDAC. Co-inhibition of DNMT and HDAC activates immune response by inducing inverted repeat Alu expression, reducing ADAR1-mediated Alu RNA editing and inducing cell type-specific TE-chimeric transcript expression. Collectively, our study demonstrates that inhibition of different CMEs results in derepression of distinct TEs in cell type-specific manner and by utilizing distinct mechanistic pathways, providing insights for epigenetic therapies that could selectively enhance anti-tumor immunity in distinct cancer types.

cancer biology↗

bioRxiv2022

Lineage-specific oncogenes drive growth of major forms of human cancer using common downstream mechanisms

Mutations in hundreds of genes have been associated with formation of human cancer, with different oncogenic lesions prevalent in different cancer types. Yet, the malignant phenotype is simple, characterized by unrestricted growth of cells that invade neighboring healthy tissue and in many cases metastasize to distant organs. One possible hypothesis explaining this dichotomy is that the cancer genes regulate a common set of target genes, which then function as master regulators of essential cancer phenotypes, such as growth, invasion and metastasis. To identify mechanisms that drive the most fundamental feature shared by all tumors - unrestricted cell proliferation - we used a multiomic approach to identify common transcriptional and posttranslational targets of major oncogenic pathways active in different cancer types, and combined this analysis with known regulators of the cell cycle. We identified translation and ribosome biogenesis as common targets of both transcriptional and posttranslational oncogenic pathways. By combining proteomic analysis of clinical samples with functional studies of cell cultures, we also establish NOLC1 as a key node whose convergent regulation both at transcriptional and posttranslational level is critical for tumor cell proliferation. Our results indicate that lineage-specific oncogenic pathways commonly regulate the same set of targets important for growth control, revealing novel key downstream nodes that could be targeted for cancer therapy or chemoprevention.

cancer biology↗

bioRxiv2021

Competitive precision genome editing (CGE) assay for functional analysis at single base resolution

MYC is an oncogenic transcription factor that controls major pathways promoting cell growth and proliferation. MYC has been implicated in the regulation of large number of genes, but the exact target genes responsible for its proliferative effects are still not known. Here, we use a novel competitive genome editing (CGE) assay for studying the functional consequence of precise mutations of MYC binding sites on cell proliferation. The CGE method is based on precision genome editing, where a CRISPR/Cas9-induced DNA break is repaired using a template that either reconstitutes the original feature or introduces an altered sequence. Both types of repair templates harbor sequence tags that allow direct comparison between cells that carry original and mutant features and generate a large number of replicate cultures. The CGE method overcomes the limitations of CRISPR/Cas9-technology in analyzing the effect of genotype on phenotype, namely the difficulty of cutting DNA exactly at the intended site, and the decreased cell proliferation caused by the DNA cuts themselves. Importantly, it provides a powerful method for studying subtle effects elicited by mutation of individual transcription factor binding sites. We show here that E-box mutations at several MYC target gene promoters resulted in reduced cellular fitness, demonstrating a direct correlation between MYC-regulated cellular processes and MYC binding and identifying important transcriptional targets responsible for its functions.

genomics↗

bioRxiv2021

Sequence determinants of human gene regulatory elements

DNA determines where and when genes are expressed, but the full set of sequence determinants that control gene expression is not known. To obtain a global and unbiased view of the relative importance of different sequence determinants in gene expression, we measured transcriptional activity of DNA sequences that are in aggregate [~]100 times longer than the human genome in three different cell types. We show that enhancers can be classified to three main types: classical enhancers1, closed chromatin enhancers and chromatin-dependent enhancers, which act via different mechanisms and differ in motif content. Transcription factors (TFs) act generally in an additive manner with weak grammar, with classical enhancers increasing expression from promoters by a mechanism that does not involve specific TF-TF interactions. Few TFs are strongly active in a cell, with most activities similar between cell types. Chromatin-dependent enhancers are enriched in forkhead motifs, whereas classical enhancers contain motifs for TFs with strong transactivator domains such as ETS and bZIP; these motifs are also found at transcription start site (TSS)-proximal positions. However, some TFs, such as NRF1 only activate transcription when placed close to the TSS, and others such as YY1 display positional preference with respect to the TSS. TFs can thus be classified into four non-exclusive subtypes based on their transcriptional activity: chromatin opening, enhancing, promoting and TSS determining factors - consistent with the view that the binding motif is the only atomic unit of gene expression.

genomics↗

bioRxiv2019

Direct conversion of human fibroblasts to liver cancer cells

Cancer is the most complex genetic disease known, with mutations implicated in more than 250 genes. However, it is still elusive which specific mutations found in human patients lead to tumorigenesis. Here we show that a combination of oncogenes that is characteristic of liver cancer (CTNNB1, TERT, MYC) induces senescence in human fibroblasts and primary hepatocytes. However, reprogramming fibroblasts to a liver progenitor fate, induced hepatocytes (iHeps), makes them sensitive to transformation by the same oncogenes. The transformed iHeps are highly proliferative, tumorigenic in nude mice, and bear gene expression signatures of liver cancer. These results show that tumorigenesis is triggered by a combination of three elements: the set of driver mutations, the cellular lineage, and the state of differentiation of the cells along the lineage. Our results provide direct support for the role of cell identity as a key determinant in transformation, and establish a paradigm for studying the dynamic role of oncogenic drivers in human tumorigenesis.

cancer biology↗