Inferring Single-Cell 3D Chromosomal Structures Based on the Lennard-Jones Potential

Reconstructing three-dimensional (3D) chromosomal structures based on single-cell Hi-C data is a challenging scientific problem due to the extreme sparseness of the single-cell Hi-C data. In this research, we used the Lennard-Jones potential to reconstruct both 500 kb and high-resolution 50 kb chromosomal structures based on single-cell Hi-C data. A chromosome was represented by a string of 500 kb or 50 kb DNA beads and put into a 3D cubic lattice for simulations. A 2D Gaussian function was used to impute the sparse single-cell Hi-C contact matrices. We designed a novel loss function based on the Lennard-Jones potential, in which the ε value, i.e., the well depth, was used to indicate how stable the binding of every pair of beads is. For the bead pairs that have single-cell Hi-C contacts and their neighboring bead pairs, the loss function assigns them stronger binding stability. The Metropolis–Hastings algorithm was used to try different locations for the DNA beads, and simulated annealing was used to optimize the loss function. We proved the correctness and validness of the reconstructed 3D structures by evaluating the models according to multiple criteria and comparing the models with 3D-FISH data.

Nuclear Disposition of Alien Chromosome Introgressions into Wheat and Rye Using 3D-FISH

During interphase, the chromosomes of eukaryotes decondense and they occupy distinct regions of the nucleus, called chromosome domains or chromosome territories (CTs). In plants, the Rabl’s configuration, with telomeres at one pole of nucleus and centromeres at the other, appears to be common, at least in plants with large genomes. It is unclear whether individual chromosomes of plants adopt defined, genetically determined addresses within the nucleus, as is the case in mammals. In this study, the nuclear disposition of alien rye and barley chromosomes and chromosome arm introgressions into wheat while using 3D-FISH in various somatic tissues was analyzed. All of the introgressed chromosomes showed Rabl’s orientation, but their relative positions in the nuclei were less clear. While in most cases pairs of introgressed chromosomes occupied discrete positions, their association (proximity) along their entire lengths was rare, and partial association only marginally more frequent. This arrangement is relatively stable in various tissues and during various stages of the cell cycle. On the other hand, the length of a chromosome arm appears to play a role in its positioning in a nucleus: shorter chromosomes or chromosome arms tend to be located closer to the centre of the nucleus, while longer arms are more often positioned at the nuclear periphery.

Instability of Alien Chromosome Introgressions in Wheat Associated with Improper Positioning in the Nucleus

Alien introgressions introduce beneficial alleles into existing crops and hence, are widely used in plant breeding. Generally, introgressed alien chromosomes show reduced meiotic pairing relative to the host genome, and may be eliminated over generations. Reduced pairing appears to result from a failure of some telomeres of alien chromosomes to incorporate into the leptotene bouquet at the onset of meiosis, thereby preventing chiasmate pairing. In this study, we analysed somatic nuclei of rye introgressions in wheat using 3D-FISH and found that while introgressed rye chromosomes or chromosome arms occupied discrete positions in the Rabl’s orientation similar to chromosomes of the wheat host, their telomeres frequently occupied positions away from the nuclear periphery. The frequencies of such abnormal telomere positioning were similar to the frequencies of out-of-bouquet telomere positioning at leptotene, and of pairing failure at metaphase I. This study indicates that improper positioning of alien chromosomes that leads to reduced pairing is not a strictly meiotic event but rather a consequence of a more systemic problem. Improper positioning in the nuclei probably impacts the ability of introgressed chromosomes to migrate into the telomere bouquet at the onset of meiosis, preventing synapsis and chiasma establishment, and leading to their gradual elimination over generations.

Aberrant Expression of the SOX11 Oncogene in Mantle Cell Lymphoma Is Associated with Activation and De Novo 3D Looping of a Distant Enhancer Element

Introduction SOX11 is a transcription factor (TF) aberrantly expressed in the majority of mantle cell lymphomas (MCLs), which is generally associated with aggressive clinical behaviour. No mutations, genetic aberrations or direct correlations with differential DNA methylation at the promoter related to its expression have been found in MCL. Deeper insights into its regulation can be found by considering the three-dimensional (3D) chromatin structure. It is becoming clear that the genome can be partitioned into 3D building blocks, topologically associated domains (TADs) and that enhancer regions likely regulate genes within their TADs by 3D contacts, but do not affect genes outside their own TADs. By mapping the 3D chromatin structure, we previously identified a distant putative SOX11 enhancer showing enhancer activity and 3D contacts with the SOX11 gene in the SOX11-positive MCL cell line Z-138, but not in the SOX11-negative MCL cell line JVM-2. Aims We aimed to deepen our understanding of the differential 3D contacts and enhancer activity previously observed at the putative SOX11 enhancer in SOX11-positive versus SOX11-negative MCL cell lines by addressing the following questions: (i) Do TAD boundaries around the SOX11 locus change between SOX11-positive and -negative MCLs? (ii) How do the 3D contacts and chromatin states at this region behave in primary MCL cases and normal B cells? (iii) Is the putative SOX11 enhancer involved in SOX11 expression in other tissues? Methods We have extended our experimental analyses of the putative SOX11 enhancer by performing (i) HiC-sequencing and 3D fluorescence in situ hybridization (3D FISH) in MCL cell lines Z-138 and JVM-2, (ii) 4C-sequencing, chromatin inmmunoprecipiation followed by deep sequencing (ChIP-seq) of 6 histone marks, an Assay for Transposase-Accessible Chromatin with deep sequencing (ATAC-seq) and chromatin state modeling by chromHMM (using the 6 histone marks) in primary MCL cases and normal naive and memory B-cells. Furthermore, we have explored the activity of this region in other SOX11 expressing cell lines studied within the ENCODE Consortium. Results HiC-sequencing in the cell lines Z-138 (SOX11-positive) and JVM-2 (SOX11-negative) showed that the SOX11 locus and its putative enhancer are located within the same TAD in both samples. Hence, shifts in TAD boundaries do not seem to underlie the differential 3D chromatin interactions between the SOX11 locus and its putative enhancer in these two cell lines. By ChIP-seq and chromatin state modeling we observed that the promoter of SOX11 is poised, i.e., carrying histone marks H3K4me3 and H3K27me3, in normal naive and memory B-cells and the SOX11-negative MCL primary case. Furthermore, we observed weak enhancer activity at the putative SOX11 enhancer in normal naive and memory B-cells and the SOX11-negative MCL primary case, but strong enhancer activity, marked by the presence of H3K27ac, only in SOX11-positive samples. In addition, by ATAC-seq we identified two specific chromatin accessible regions that potentially represent the transcription factor binding sites responsible for activation of this enhancer region in SOX11-positive MCLs. By 4C-sequencing we observed that the SOX11 locus and its putative enhancer show high 3D contacts in two other SOX11-positive MCL cell lines (GRANTA-519 and JEKO-1) and in a SOX11-positive primary MCL case, but not in a SOX11-negative primary MCL case. Furthermore, the differential 3D contacts at these regions in Z-138 and JVM-2 were confirmed by 3D FISH, which is currently being performed in primary MCL cases. Interestingly, no 3D contacts were observed in normal naive and memory B cells, indicating that although the SOX11 promoter is poised within these normal B-cell subpopulations, primed looping at these regions does not exist and seems not to explain the 3D contacts we observed in SOX11-positive MCL cell lines and primary cases. When investigating chromatin states in cell lines studied by ENCODE with an active SOX11 promoter (H1-hESC, HSMM and NHLF) none of them show activity in the identified region, suggesting that the putative SOX11 enhancer is de novo activated only in the context of MCL lymphomagenesis. Conclusions We provide new evidence that the activation of a distant SOX11 putative enhancer and its 3D contacts to the SOX11 gene, is a de novo event in SOX11-positive MCL cell lines and primary cases that is likely specific for this malignancy. Disclosures No relevant conflicts of interest to declare.