Thousands of long non-coding RNAs (lncRNAs) have been identified in mammals, many of which represent important regulators of gene expression. However, the mechanisms used by lncRNAs to control transcription remain largely uncharacterized. Here, we report on HOTAIRM1, a promising lncRNA biomarker in leukemia and solid tumors. We find that HOTAIRM1 contributes to three-dimensional chromatin organization changes required for the temporal collinear activation of HOXA genes. We show that distinct HOTAIRM1 variants preferentially associate with either UTX/MLL or PRC2 complexes to modulate the levels of activating and silencing marks at the bivalent domain. HOTAIRM1 contributes to physical dissociation of chromatin loops at the cluster proximal end, which delays recruitment of the histone demethylase UTX and transcription of central HOXA genes. Interestingly, we find overall proximal HOXA gene activation without chromatin conformation changes by HOTAIRM1 in a different cell type. Our results reveal a previously unappreciated relationship between chromatin structure, architecture and lncRNA function.

Several types of collagen contain cryptic antiangiogenic noncollagenous domains that are released upon proteolysis of extracellular matrix (ECM). Among those is Arresten, a collagen-derived antiangiogenic factor (CDAF) that is processed from a1 collagen IV. However, the conditions under which Arresten is released from collagen IV in vivo or whether the protein functions in tumor suppressor pathways remain unknown. Here, we show that p53 induces the expression of a1 collagen IV and release of Arresten-containing fragments from the ECM. Comparison of the transcriptional activation of COL4A1 with other CDAF-containing genes revealed that COL4A1 is a major antiangiogenic gene induced by p53 in human adenocarinoma cells. p53 directly activated transcription of the COL4A1 gene by binding to an enhancer region 26 kbp downstream of its 3 0 end. p53 also stabilized the expression of full-length a1 collagen IV by upregulation of a(II) prolyl-hydroxylase and increased the release of Arresten in the ECM through a matrix metalloproteinase (MMP)-dependent mechanism. The resulting upregulation of a1 collagen IV and production of Arresten by the tumor cells significantly inhibited angiogenesis and limited tumor growth in vivo. Furthermore, we show that immunostaining of Arresten correlated with p53 status in human prostate cancer specimens. Our findings, therefore, link the production of Arresten to the p53 tumor suppressor pathway and show a novel mechanism through which p53 can inhibit angiogenesis. Cancer Res; 72(5);

Objective: Ligation-Mediated Amplification (LMA) is a versatile biochemical tool for amplifying selected DNA sequences. LMA has increased in popularity due to its integration within chromosome conformation capture (5C) and chromatin immunoprecipitation (2C-ChIP) methodologies. The output of either 5C or 2C-ChIP protocols is a single-read sequencing library of ligated primer pairs that may or may not be multiplexed. While many computational tools currently exist for read mapping and analysis, these tools neither fully support multiplexed libraries nor provide qualitative reporting on the LMA primers involved. Typically, the task of library demultiplexing or primer analysis is offloaded on to the user. Our aim was to develop an easy-to-use pipeline for processing (multiplexed) single-read sequencing data produced by sequence-specific LMA. Results: Here, we describe the Ligation-mediated Amplified, Multiplexed Primer-pair Sequence (LAMPS) analysis pipeline. LAMPS facilitates the analysis of multiplexed LMA sequencing data and provides a thorough assessment of a library's reads for a variety of experimental parameters (e.g., primer-pair efficiency). The standardized output of LAMPS allows for easy integration with downstream analyses, such as data track visualization on a genome browser.