Expanding the scope of methylation-sensitive restriction enzyme (MSRE) PCR for forensic identification of body fluids through the novel use of methylation-dependent restriction enzymes (MDRE) and the combination of autosomal and Y-chromosomal markers.

Methylation-sensitive/-dependent restriction enzyme (MSRE/MDRE) PCR can be performed to detect hypomethylated or hypermethylated CpG sites. With the combined use of different tissue-specific CpG markers, MSRE/MDRE-PCR leads to tissue-specific methylation patterns (TSMPs), enabling the correlation of DNA samples to their source tissue. MSRE/MDRE assays can use the same platform as forensic STR typing and offer many advantages in the field of forensic body fluid detection. In the present study, we aimed to establish MSRE assays for the detection of blood, saliva, vaginal secretion, and semen, using markers from literature and from our own database search. We designed two different MSRE test-sets, which include two novel Y-chromosomal non-semen markers, and enable differentiation between female and male non-semen samples. Furthermore, we established an MSRE/MDRE semen approach, which includes only Y-chromosomal non-semen and semen markers. This Y-semen multiplex PCR utilizes the novel combination of the methylation-sensitive enzyme SmaI and the methylation-dependent enzyme GlaI, which enables more sensitive detection of male body fluids within male/female DNA mixtures. Our validation tests confirmed that MSRE/MDRE assays exhibit high sensitivity, similar to that of STR typing.

Fluid shear stress-modulated chromatin accessibility reveals the mechano-dependency of endothelial SMAD1/5-mediated gene transcription.

Bone morphogenetic protein (BMP) signaling and fluid shear stress (FSS) mediate complementary functions in vascular homeostasis and disease development. It remains to be shown whether altered chromatin accessibility downstream of BMP and FSS offers a crosstalk level to explain changes in SMAD-dependent transcription. Here, we employed ATAC-seq to analyze arterial endothelial cells stimulated with BMP9 and/or FSS. We found that BMP9-sensitive regions harbor non-palindromic GC-rich SMAD-binding elements (GGCTCC) and 69.7% of these regions become BMP-insensitive in the presence of FSS. While GATA and KLF transcription factor (TF) motifs are unique to BMP9- and FSS-sensitive regions, respectively, SOX motifs are common to both. Finally, we show that both SOX(13/18) and GATA(2/3/6) family members are directly upregulated by SMAD1/5. These findings highlight the mechano-dependency of SMAD-signaling by a sequential mechanism of first elevated pioneer TF expression, allowing subsequent chromatin opening to eventually providing accessibility to novel SMAD binding sites.