The tardigrade Hypsibius exemplaris dramatically upregulates DNA repair pathway genes in response to ionizing radiation.

Tardigrades can survive remarkable doses of ionizing radiation, up to about 1,000 times the lethal dose for humans. How they do so is incompletely understood. We found that the tardigrade Hypsibius exemplaris suffers DNA damage upon gamma irradiation, but the damage is repaired. We show that this species has a specific and robust response to ionizing radiation: irradiation induces a rapid upregulation of many DNA repair genes. This upregulation is unexpectedly extreme-making some DNA repair transcripts among the most abundant transcripts in the animal. By expressing tardigrade genes in bacteria, we validate that increased expression of some repair genes can suffice to increase radiation tolerance. We show that at least one such gene is important in vivo for tardigrade radiation tolerance. We hypothesize that the tardigrades' ability to sense ionizing radiation and massively upregulate specific DNA repair pathway genes may represent an evolved solution for maintaining DNA integrity.

Balance between immunoregulatory B cells and plasma cells drives pancreatic tumor immunity.

Plasma cell responses are associated with anti-tumor immunity and favorable response to immunotherapy. B cells can amplify anti-tumor immune responses through antibody production; yet B cells in patients and tumor-bearing mice often fail to support this effector function. We identify dysregulated transcriptional program in B cells that disrupts differentiation of naive B cells into anti-tumor plasma cells. The signaling network contributing to this dysfunction is driven by interleukin (IL) 35 stimulation of a STAT3-PAX5 complex that upregulates the transcriptional regulator BCL6 in naive B cells. Transient inhibition of BCL6 in tumor-educated naive B cells is sufficient to reverse the dysfunction in B cell differentiation, stimulating the intra-tumoral accumulation of plasma cells and effector T cells and rendering pancreatic tumors sensitive to anti-programmed cell death protein 1 (PD-1) blockade. Our findings argue that B cell effector dysfunction in cancer can be due to an active systemic suppression program that can be targeted to synergize with T cell-directed immunotherapy.

Adaptation and selection shape clonal evolution of tumors during residual disease and recurrence.

The survival and recurrence of residual tumor cells following therapy constitutes one of the biggest obstacles to obtaining cures in breast cancer, but it remains unclear how the clonal composition of tumors changes during relapse. We use cellular barcoding to monitor clonal dynamics during tumor recurrence in vivo. We find that clonal diversity decreases during tumor regression, residual disease, and recurrence. The recurrence of dormant residual cells follows several distinct routes. Approximately half of the recurrent tumors exhibit clonal dominance with a small number of subclones comprising the vast majority of the tumor; these clonal recurrences are frequently dependent upon Met gene amplification. A second group of recurrent tumors comprises thousands of subclones, has a clonal architecture similar to primary tumors, and is dependent upon the Jak/Stat pathway. Thus the regrowth of dormant tumors proceeds via multiple routes, producing recurrent tumors with distinct clonal composition, genetic alterations, and drug sensitivities.

Multigenome DNA sequence conservation identifies Hox cis-regulatory elements.

To learn how well ungapped sequence comparisons of multiple species can predict cis-regulatory elements in Caenorhabditis elegans, we made such predictions across the large, complex ceh-13/lin-39 locus and tested them transgenically. We also examined how prediction quality varied with different genomes and parameters in our comparisons. Specifically, we sequenced approximately 0.5% of the C. brenneri and C. sp. 3 PS1010 genomes, and compared five Caenorhabditis genomes (C. elegans, C. briggsae, C. brenneri, C. remanei, and C. sp. 3 PS1010) to find regulatory elements in 22.8 kb of noncoding sequence from the ceh-13/lin-39 Hox subcluster. We developed the MUSSA program to find ungapped DNA sequences with N-way transitive conservation, applied it to the ceh-13/lin-39 locus, and transgenically assayed 21 regions with both high and low degrees of conservation. This identified 10 functional regulatory elements whose activities matched known ceh-13/lin-39 expression, with 100% specificity and a 77% recovery rate. One element was so well conserved that a similar mouse Hox cluster sequence recapitulated the native nematode expression pattern when tested in worms. Our findings suggest that ungapped sequence comparisons can predict regulatory elements genome-wide.

A highly conserved molecular switch binds MSY-3 to regulate myogenin repression in postnatal muscle.

Myogenin is the dominant transcriptional regulator of embryonic and fetal muscle differentiation and during maturation is profoundly down-regulated. We show that a highly conserved 17-bp DNA cis-acting sequence element located upstream of the myogenin promoter (myogHCE) is essential for postnatal repression of myogenin in transgenic animals. We present multiple lines of evidence supporting the idea that repression is mediated by the Y-box protein MSY-3. Electroporation in vivo shows that myogHCE and MSY-3 are required for postnatal repression. We further show that, in the C2C12 cell culture system, ectopic MSY-3 can repress differentiation, while reduced MSY-3 promotes premature differentiation. MSY-3 binds myogHCE simultaneously with the homeodomain protein Pbx in postnatal innervated muscle. We therefore propose a model in which the myogHCE motif operates as a switch by specifying opposing functions; one that was shown previously is regulated by MyoD and Pbx and it specifies a chromatin opening, gene-activating function at the time myoblasts begin to differentiate; the other includes MYS-3 and Pbx, and it specifies a repression function that operates during and after postnatal muscle maturation in vivo and in myoblasts before they begin to differentiate.

New computational approaches for analysis of cis-regulatory networks.

The investigation and modeling of gene regulatory networks requires computational tools specific to the task. We present several locally developed software tools that have been used in support of our ongoing research into the embryogenesis of the sea urchin. These tools are especially well suited to iterative refinement of models through experimental and computational investigation. They include: BioArray, a macroarray spot processing program; SUGAR, a system to display and correlate large-BAC sequence analyses; SeqComp and FamilyRelations, programs for comparative sequence analysis; and NetBuilder, an environment for creating and analyzing models of gene networks. We also present an overview of the process used to build our model of the Strongylocentrotus purpuratus endomesoderm gene network. Several of the tools discussed in this paper are still in active development and some are available as open source.